Microsystems for in-vitro Cell Models

In-vitro cell models are used for disease modelling, for screening active substances and for toxicity tests, as well as for fundamental research into the development and organization of diverse types of tissue. Based on our expertise in the fields of cell biology, material sciences and coatings, as well as microsensor systems and microfluidics (design, production and integration), we develop microreactors for short-term and long-term studies on cell aggregates. These reactors allow cells to be cultured under controlled physiological conditions. The integration of microsensors into these systems allows, for the very first time, the continual measurement of cell vitality or metabolites for up to one month in real time. In comparison to conventional endpoint analyses, this approach provides far more immediate access to an understanding of the mechanisms of action. Along with concept creation and the development of micro-bioreactors, we also conduct in-vitro analyses to assess the hepatotoxicity of chemical substances.

Microfluidic Bioreactor for Hepatocytes

Microfluidic Bioreactor for Hepatocytes
© Fraunhofer IZI-BB

Micro-bioreactor for hepatocytes for the assessment of the toxicity of chemicals, such as active substances. The metabolic activity of the cells can be analyzed in real time over a period of several weeks in the reactor. To do so, oxygen-sensitive microparticles (top left) are deposited into small cavities together with hepatocytes (top right). The particles are easy to sort out by optical means. The graph (bottom left) clearly indicates two mechanisms of action of the painkiller acetaminophen (paracetamol) which can be distinguished by their kinetics.

The diagram shows fully automated specimen removal from a micro-bioreactor (in red) for regular determination of the glucose and lactate concentrations. This information allows the metabolic activity of hepatocytes to be evaluated. This provides an important foundation for the development of in-vitro assays for the evaluation of the toxicity of active substances.
© Fraunhofer IZI-BB

The diagram shows fully automated specimen removal from a micro-bioreactor (in red) for regular determination of the glucose and lactate concentrations. This information allows the metabolic activity of hepatocytes to be evaluated. This provides an important foundation for the development of in-vitro assays for the evaluation of the toxicity of active substances.

The institute develops in-vitro test procedures for the assessment of the long-term toxicity of active substances, in order to replace medium-term animal testing. Maintaining the vitality of cell cultures over sufficiently long periods requires continual monitoring of the cultivation conditions. The concentration of glucose and oxygen, along with the pH level of the cell culture medium in the bioreactor, are the key parameters for this process. The continual measurement of these variables not only permits rigorous quality assurance, but also provides the input signals for automated microreactor operation. A significant number of the activities pursued by the Working Group is devoted to the development of sensor technology, and its integration into the microreactors. This presents challenges associated with miniaturization, and which are therefore related to the minute volumes of specimens available, as well as the requirements of maintaining long-term stability.

Methods

  • Development and production of functional coatings for applications in the field of cell cultivation and tissue engineering: Coatings made of thermoresponsive polymers for monitoring cell adhesion on cell culture substrates, polyelectrolyte layers (layer-by-layer (LbL) application) and reservoirs for biomolecules for controlling adherent cells, layers (self-assembled monolayers (SAM)) made of polymers, and biomolecules for improving the biocompatibility of synthetic surfaces
  • Design and development of micro-bioreactors for the long-term cultivation of complex cell models
  • Integration of microsensors into microfluidic systems for real-time analysis of key variables of cell media (e.g. oxygen, pH, glucose, lactate)
  • Development of in-vitro test systems for the assessment of the toxicity of chemicals, pharmaceutical agents and ingredients used in cosmetics
  • Storage and cultivation of eukaryotic cells at S1 biosafety level (mammalian cells, insect cells, primary cells, cell lines)
     

Equipment

  • Transmitted and reflected light microscopy with bright field, phase contrast, fluorescence, polarization and total reflection modes (TIRFM), super-resolution structured illumination microscopy (SIM), each equipped with computer-controlled and temperature-controlled specimen stages and cell culture chambers
  • Confocal scanning laser microscope with 3D image processing
  • Fully automated fluorescence microscope for producing images of living cells under physiological conditions (time-lapse microscopy) (Olympus CellR)
  • TIRF microscopy (Olympus)
  • Laser tweezers with laser microdissection (Palm/Zeiss)
  • Variable microfluidics setup
  • Microcontact printer (GeSiM)
  • Contact angle measuring device
  • Flow cytometer (Becton D.)
  • Micromanipulation, microinjection, microdissection (Eppendorf)
  • Cell characterization: Cell staining techniques (e.g. immunofluorescence), transfection with fluorescent fusion proteins, live staining, proliferation tests

  • GeSiM mbH, Grosserkmannsdorf
  • Mikrofluidik ChipShop, Jena
  • BST Bio Sensor Technology GmbH
  • University of Jerusalem, Israel
  • École Polytechnique Fédéral de Lausanne, Switzerland
  • Centre Suisse d’Electronique et Microtechnique Neuchâtel, Switzerland
  • University of Bielefeld
  • Nottingham Trent University

Publications

  • 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.
  • Prill S, Jaeger, MS, Duschl C. Long-term microfluidic glucose and lactate monitoring in hepatic cell culture. Biomicrofluidics. (2014) 8, 034102.
  • 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, 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.
  • 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
  • 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