Microsystems for In Vitro Cell Models

This unit offers the application-related and customer-specific development of procedures and prototypes for cultivating, characterizing and processing demanding cell samples. Our expertise in microreactors, microfluidics, sensor technology and functional polymer coatings forms the basis of innovative solution concepts, which are complemented by our established knowledge in the fields of cell biology, toxicology and bioanalytics. The unit's interdisciplinary orientation enables us to provide well-founded, targeted advice and to efficiently cater to your specific needs. Our work focuses on (i) developing in-vitro test procedures to be able to assess the toxicity of drugs and chemicals based on highly functional microbioreactors and sophisticated cell models, as well as (ii) establishing intelligent polymer coatings which allow the behavior of adherent cells to be controlled on technical surfaces. State-of-the-art facilities are available for all of these development tasks.

  • Design and development of microbioreactors for the long-term cultivation of sophisticated cell models
  • Integration of microsensors into microfluidic systems for the real-team recording of cell media parameters (e.g. oxygen, pH, glucose, lactate)
  • Development of in-vitro test systems to assess the toxicity of chemicals, pharmaceutical drugs and cosmetics components
  • Development and production of functional coatings for applications in the field of cell cultivation and tissue engineering: Coatings made from thermoresponsive polymers to monitor cell adhesion on cell culture substrates; polyelectrolyte layers (layer-by-layer (LbL) application) as reservoirs for biomolecules to control adherent cells; layers (self assembled monolayers (SAM)) made from polymers and biomolecules to improve the biocompatibility of synthetic surfaces
  • Techniques for manufacturing homogeneous and structured coatings: spin coating, dip coating, spraying, spotting, printing (µ-contact printing)
  • Broad spectrum of methods for the noninvasive examination and characterization of surfaces and coatings: contact-angle determination, ellipsometry, surface plasmon resonance (SPR), fluorescence microscopy techniques, fluorescence recovery after photobleaching (FRAP), atomic force microscopy (AFM)
  • Time-resolved examination of cell adhesion on functionalized surfaces using total internal reflection fluorescence microscopy (TIRFM)
  • Characterization of the mechanical properties of surfaces and coatings by means of microindentation
  • Storage and cultivation of eukaryotic cells at S1 level (mammalian cells, insect cells, primary cells, cell lines)

Microfluidic bioreactor for hepatocytes

Microbioreactor for hepatic cells to assess the toxicity of chemicals, e.g. active ingredients. The metabolic activity of cells can be measured in real time in the reactor over a period of several weeks. In order to do this, oxygen-sensitive microparticles (top left) are deposited in small cavities (top right) together with hepatic cells. The particles can simply be selected optically. The graph (bottom left) clearly shows two mechanisms of action for the pain reliever acetaminophen (paracetamol) which can be differentiated based on their kinetics.
© Photo Fraunhofer IZI

Microbioreactor for hepatic cells to assess the toxicity of chemicals, e.g. active ingredients. The metabolic activity of cells can be measured in real time in the reactor over a period of several weeks. In order to do this, oxygen-sensitive microparticles (top left) are deposited in small cavities (top right) together with hepatic cells. The particles can simply be selected optically. The graph (bottom left) clearly shows two mechanisms of action for the pain reliever acetaminophen (paracetamol) which can be differentiated based on their kinetics.

The diagram shows fully automated sampling from a microbioreactor (in red) for the regular determination of glucose and lactate concentrations. The metabolic activity of hepatic cells can be evaluated using this information. This provides an important foundation for the development of in-vitro assays that can be used to assess drug toxicity.
© Photo Fraunhofer IZI

The diagram shows fully automated sampling from a microbioreactor (in red) for the regular determination of glucose and lactate concentrations. The metabolic activity of hepatic cells can be evaluated using this information. This provides an important foundation for the development of in-vitro assays that can be used to assess drug toxicity.

This unit develops in-vitro test methods for evaluating the long-term toxicity of active ingredients with the aim of replacing animal experiments in the medium term. Preserving the vitality of cell cultures for sufficiently long periods of time requires the constant monitoring of cultivation conditions. The concentrations of glucose and oxygen as well as the pH value of the cell culture medium in the bioreactor are the most important parameters here. The continuous measurement of these parameters not only permits rigorous quality control, but also delivers the input signals for the automated operation of the microreactor. A significant part of our activities is therefore dedicated to the development of sensor technology and its integration in the microreactors. The challenges come from the miniaturization and are associated with the tiny available sample volume as well as the requirements for long-term stability.

Thermoresponsive polymer coatings to control cell adhesion

Controlling cell adhesion on thermoresponsive surfaces. At 37°C, the cells are adherent and spread out over the surfaces. If the surface temperature drops to 25°C, the cells break away from their substrate and can be removed simply by rinsing.
© Photo Fraunhofer IZI

Controlling cell adhesion on thermoresponsive surfaces. At 37°C, the cells are adherent and spread out over the surfaces. If the surface temperature drops to 25°C, the cells break away from their substrate and can be removed simply by rinsing.

Controlling cell adhesion on structured thermoresponsive surfaces. Thermoresponsive microgels can be applied locally to many surfaces (in this case forming circular islands). Homogeneous cells develop at 37°C (1). Upon reducing the surface temperature to 25°C, the cells selectively break away from their substrate (2 & 3). If the temperature is increased once more, the cells repopulate the thermoresponsive areas (4).
© Photo Fraunhofer IZI

Controlling cell adhesion on structured thermoresponsive surfaces. Thermoresponsive microgels can be applied locally to many surfaces (in this case forming circular islands). Homogeneous cells develop at 37°C (1). Upon reducing the surface temperature to 25°C, the cells selectively break away from their substrate (2 & 3). If the temperature is increased once more, the cells repopulate the thermoresponsive areas (4).

This unit develops thermoresponsive polymer coatings on which cells demonstrate good adhesion properties at typical cultivation temperatures. If the temperature is reduced by just a few degrees, the cells can be detached from these coatings simply by rinsing, without the need for invasive enzyme cocktails. It is thus ensured that the vitality of the cells is not impaired during this often critical stage in the process. The polymers can be applied to almost all usual cell culture substrates, cost-effectively, either homogenously or in defined patterns using simple methods such as spin coating, spotting or printing. The application area for thermoresponsive coatings can therefore be expanded far beyond the careful performance of standard protocols. Cocultures with defined geometric relationships can thus be manufactured easily using this approach. Tests show that cell assays, e.g. for wound repair or cell migration, can be handled much better using the polymer coatings, while reliability and precision are also improved. Establishing novel cell assay formats forms another focal area within the unit.

By reducing the cultivation temperature from 37°C to 28°C, a thermoresponsive cell cultivation substrate goes from a cell-attracting to a cell-repellent state. This leads to the noninvasive detachment of the cells from the substrate.

Depiction of the contact area of fibroblasts on a thermoresponsive cell cultivation substrate during cell detachment.

  • Transmitted-light and reflected-light microscopy with brightfield, phase contrast, fluorescence, polarization and total reflection mode (TIRFM), high-resolution optical microscopy (SIM), each equipped with computer-controlled and tempered object plates and cell culture chambers
  • Confocal scanning laser microscope with 3D image processing
  • Fully automated fluorescence microscopes for recording living cells under physiological conditions (time-lapse microscopy) (Olympus CellR)
  • TIRF microscopy (Olympus)
  • Laser tweezers / optical tweezers with laser micro-dissection (Palm / Zeiss)
  • Atomic force microscopy for biological applications / Bio-AFM (JPK)
  • Variable microfluidics setup
  • Laboratory for surface chemistry and biochemistry
  • Multiskop for imaging ellipsometry, surface plasmon resonance / SPR (Optrel)
  • Evaporation coater for manufacturing thin metallic layers (Edwards)
  • Microcontact printer (GeSiM)
  • Contact-angle meter
  • 200 m² fully equipped cell cultivation area
  • Flow cytometer (Becton D.)
  • High-throughput plate reader (CellLux)
  • Micromanipulation, micro-injection, microdissection (Eppendorf)
  • Cryopreservation of cells
  • Cell characterization: cell staining techniques (e.g. immunofluorescence), transfection with fluorescent fusion proteins, intravital staining, proliferation tests

  • GeSiM mbH, Großerkmannsdorf
  • Mikrofluidik ChipShop, Jena
  • BST Bio Sensor Technology GmbH
  • University of Jerusalem, Israel
  • Ecole Polytechnique Federal Suisse, Lausanne, Switzerland
  • Centre Suisse dElectronique et Microtechnique Neuchâtel, Switzerland
  • Universität Bielefeld
  • Nottingham Trent University

  • Bavli D, Prill P, Ezra E, Levy G, Cohen M, Vinken M, Vanfleteren J, Jaeger MS, Nahmias Y. Real-time monitoring of metabolic function in liver-on-chip microdevices tracks the dynamics of mitochondrial dysfunction. PNAS (2016) 113, S. E2231-E2240.
  • Prill S, Bavli D, Jaeger MS, Schmälzlin E, Levy G, Schwarz M, Duschl C, Ezra E, Nahmias Y. A Real-Time Monitoring of Oxygen Uptake in Hepatic Microwell Bioreactor Reveals CYP450-Independent Direct Mitochondrial Toxicity of Acetaminophen multilayers. Archives of Toxicology, 90 (2016) 1181-1191. DOI dx.doi.org/10.1007/s00204-015-1537-2
  • Prokopovic VZ, Vikulina AS, Sustr D, Duschl C, Volodkin D. Towards an artificial extracellular matrix: Biopolymer based multilayers coated with gold nanoparticles. Assessment of biodegradation, molecular transport, and protein mobility. ACS Applied Materials and Interfaces 8 (2016) S. 24345-24349.
  • Uhlig K, Wegener T, He J, Zeiser M, Bookhold J, Dewald I, Godino N, Jaeger MS, Hellweg T, Fery A, Duschl C. Patterned thermoresponsive microgel coatings for noninvasive processing of adherent cells. Biomacromolecules (2016),  17, S. 1110-1116.
  • Velk N, Uhlig K, Duschl C, Volodkin D. Mobility of Lysozyme in Poly(L-lysine)/Hyaluronic Acid Multilayer Films. Colloids Surfaces B (2016), 47, S. 343-350.
  • Vikulina AS, Anissimov YG, Singh P, Prokopović VZ, Uhlig U, Jaeger MS, von Klitzing R, Duschl C, Volodkin D. Temperature effect on build-up of exponentially growing polyelectrolyte multilayers. Exponential-to-linear transition point. Phys. Chem. Chem. Phys. (2016), 18, S. 7866-7874.
  • Prokopovic VZ, Duschl C, Volodkin D. Hyaluronic acid/poly-L-lysine Multilayers as Reservoirs for Storage and Release of Small Charged Molecules. Macromo. Biosci. (2015), 15, S. 1357-1363.
  • Vikulina AS, Aleed ST, Paulraj T, Vladimirov YA, von Klitzing R, Duschl C, Volodkin D. Temperature-induced molecular transport through polymer multilayers coated with pNIPAM microgels. Phys. Chem. Chem. Phys. (2015), 17, S. 12771-12777.
  • Paulraj T, Feoktistova N, Velk N, Uhlig K, Duschl C, Volodkin D. Microporous polymeric 3D scaffolds templated by the Layer-by-Layer self-assembly. Macromol. Rapid Comm. (2014) 35, S. 1408-1413.
  • Prill S, Jaeger, MS, Duschl C. Long-term microfluidic glucose and lactate monitoring in hepatic cell culture. Biomicrofluidics. (2014) 8, 034102.
  • Schmidt S, Uhlig K, Duschl C, Volodkin D. Stability and Cell Uptake of Calcium Carbonate Templated Insulin Microparticles. Acta Biomat. (2014), 10, S. 1423-1430.
  • Uhlig K, Boerner HG, Wischerhoff E, Lutz JF, Jaeger MS, Laschewsky A, Duschl C. On the interaction of adherent cells with thermoresponsive polymer coatings. Polymers. (2014), 6, 1164-1177.
  • Renner A, Jaeger MS, Lankenau A, Duschl C. Position-dependent chemotactic response of slowly migrating cells in sigmoidal concentration profiles. Appl Phys A. (2013), 112(3), 637-645.
  • Madaboosi N, Uhlig K, Jäger MS, Möhwald H, Duschl C, Volodkin D. Microfluidics as A Tool to Understand the Build-Up Mechanism of Exponential-Like Growing Films. Macromol Rapid Comm. (2012), 33(20), 1775-1779.
  • Madaboosi N, Uhlig K, Schmidt S, Jaeger MS, Möhwald H, Duschl C, Volodkin D. Microfluidics meets soft layer-by-layer films: selective cell growth in 3D polymer architectures. Lab Chip. (2012), 12, S. 1434-1436.
  • Schmidt S, Behra M, Uhlig K, Madaboosi N, Hartmann L, Duschl C, Volodkin D. Mesoporous Protein Particles through Colloidal CaCO3 Templates. Adv. Funct. Mat. (2012) 23, S. 116-123.
  • Uhlig K, Boysen B, Lankenau A, Jaeger MS, Wischerhoff E, Lutz JF, Laschewsky A, Duschl C. On the influence of the architecture of poly(ethylene glycol)-based thermoresponsive polymers on cell adhesion. Biomicrofluidics (2012), 6, S. 024129.
  • Uhlig K, Madaboosi N, Schmidt S, Jäger MS, Rose J, Duschl C, Volodkin D. 3D localization and diffusion of proteins in polyelectrolyte multilayers. Soft Matter (2012),  8, S. 11786-11789.
  • Volodkin VS, Schmidt S, Fernandes P, Larionova NI, Sukhorukov GB, Duschl C, Möhwald H, von Klitzing R. One-step formulation of protein microparticles with tailored properties: hard templating at soft conditions. Adv. Funct. Mat. (2012), 22, S. 1914-1922.
  • Schmidt S, Zeiser M, Hellweg T, Duschl C, Fery A, Möhwald H. Adhesion and Mechanical Properties of PNIPAM Microgel Films and their Potential Use as Switchable Cell Culture Substrates. Adv. Funct. Mat. (2010), 20, S. 3235-3244.
  • Uhlig K, Wischerhoff E, Lutz JF, Laschewsky A, Jaeger MS, Lankenau A, Duschl C. Monitoring cell detachment on PEG-based thermoresponsive surfaces using TIRF microscopy. Soft Matter. (2010), 6, 4262-4267.
  • Kessel S, Müller R, Schmidt S, Wischerhoff E, Laschewsky A, Lutz JF, Uhlig K, Lankenau A, Duschl C and Fery A. Thermoresponsive, PEG-based Polymer Layers: Surface Characterization with AFM Force Measurements. Langmuir (2009), 26, S. 3462–3467.
  • Ernst O, Lieske A, Holländer A, Lankenau A, Duschl C. Tailoring of Thermo-Responsive Self-Assembled Monolayers for Cell Type Specific Control of Adhesion. Langmuir (2008), 24, S. 10259.
  • Felten M, Staroske W, Jaeger MS, Schwille P, Duschl C. Accumulation and filtering of nanoparticles in microchannels using electrohydrodynamically induced vortical flows. Electrophoresis. (2008), 29, 2987-2996.
  • Jaeger MS, Uhlig K, Clausen-Schaumann H, Duschl C. The structure and functionality of contractile forisome protein aggregates. Biomaterials. (2008), 29, 247–256.
  • Uhlig K, Jaeger MS, Lisdat F, Duschl C. A biohybrid microfluidic valve based on forisome protein complexes. J MEMS. (2008), 17(6), 1322-1328
  • Wischerhoff E, Uhlig K, Lankenau A, Börner HG, Laschewsky A, Duschl C, Lutz JF. Controlled Cell Adhesion on PEG-based Switchable Surfaces. Angew. Chem. (2008), 47, S. 5666-5668.
  • Ernst O, Lieske A, Jaeger M, Lankenau A, Duschl C. Control of cell detachment in a microfluidic device using a thermoresponsive copolymer on a gold substrate. Lab Chip. (2007), 7, 1322–1329.
  • Felten M, Geggier P, Jaeger M, Duschl C. Controlling electrohydrodynamic pumping in microchannels through defined temperature fields. Phys Fluids. (2006), 18, 051707.
  • Gast FU, Dittrich PS, Schwille P, Weigel M, Mertig M, Opitz J, Queitsch U, Diez S, Lincoln B, Wottawah F, Schinkinger S, Guck J, Käs J, Smolinski J, Salchert K, Werner C, Duschl C, Jäger M, Uhlig K, Geggier P, Howitz S. The microscopy cell (MicCell), a versatile modular flowthrough system for cell biology, biomaterial research, and nanotechnology. Microfluid Nanofluid. (2006), 2, 21–36.

Patents

  • Jaeger M, Prill S, Nahmias Y, Bavli D. Method and system for continous monitoring of toxicity. EP15160661.3 / US 2015/0268224 A1
  • Duschl C, Lankenau A, Lutz J-F, Laschewsky A, Wischerhoff E, Fuhr GR, Bier F. Substrat, Kultivierungseinrichtung und Kultivierungsverfahren für biologische Zellen. DE 10 2010 012 254 A1. 22. Sept. 2011.
  • Duschl C, Hellweg T, Lankenau A, Laschewsky A, Lutz J-F, Schmidt S, Wischerhoff E. Thermoresponsives Substrat mit Mikrogelen, Verfahren zu dessen Herstellung und Kultivierungsverfahren für biologische Zellen. DE 10 2010 012 252 A1. 22. Sept. 2011.