Microfluidic Cell Processing and Cell Analytics

This unit offers the application-related and customer-specific development of procedures and prototypes to process and manipulate demanding biological samples. It focuses in part on manipulating individual objects, e.g. the gentle and versatile handling of single cells and particularly small cell samples in microfluidic chips. This usually involves the use of electric fields in the radio frequency range. For more complicated tasks, this is combined with complementary manipulation procedures involving optical tweezers or microfluidic processes.

Besides this, the unit deals with the integration of sensor technology in microfluidic components in order to record key parameters relating to cells and other complex biological samples. State-of-the-art facilities are available for these development tasks.

  • Functional cell assays and cell analytics (e.g. chemotaxis, neurite growth, stem cell differentiation, calcium signaling, cytotoxicity, etc.)
  • Design and structuring of chip-based microsystems for the cell-compatible injection of physiological suspensions into microfluidic systems; contact-free handling of individual or low numbers of biological objects (cells, bacteria, viruses) and targeted deposition of previously characterized particles for further cultivation.
  • Design and development of microfluidic systems (chips, peripheral equipment, detection) related to biotechnology and cell biology
  • Microsystems for the controlled translation and rotation of suspended microparticles
  • Manual, semiautomatic and automatic sorting of micro-objects (e.g. living cells) in continuous flow systems
  • Washing and loading living cells without centrifugation with e.g. pharmaceutical agents in microfluidic flow systems
  • Dielectric characterization of complex particles at single cell level
  • Chip-based electromanipulation (e.g. fusion) of rare cells (e.g. stem cells)
  • Combination of dielectric trapping fields and optical tweezers to manipulate several objects at the same time and to characterize interactions (binding forces) between particles
  • High-end optical microscopy, e.g. highly photosensitive fluorescence measurements
  • Numerical calculation and modelling using the finite element method
  • Influence of alternating electric fields (10 kHz to 250 MHz) on biological objects
  • Micromanipulation of individual objects by means of capillary aspiration
  • Microprocessing using UV laser ablation
  • Cultivation of animal and yeast cultures at S1 level before and after their manipulation in microfluidic chips
  • Kinetic binding studies on receptor-ligand interactions in microchannels
  • Time-resolved fluorescence microscopy of living cell systems
  • Immunoassays for the detection of blood and plasma proteins
  • Bloodwork laboratory for hemocompatibility testing
  • Photolithographic process line
  • Microstructuring of metallized surfaces using photolithography
  • Manufacture of micro-contact stamping and establishing the stamping process

Single cell handling

Targeted contacting of a single T-cell with a functionalized microparticle in a microchannel.
© Photo Fraunhofer IZI

Targeted contacting of a single T-cell with a functionalized microparticle in a microchannel. The increase in intracellular Ca2+ concentration, triggered by the binding of the cells to the particle surface, was measured using a calcium-sensitive fluorescent dye.

This unit has years of experience when it comes to handling valuable cell samples at single cell level. With the aid of freely configurable microelectrodes, which are integrated into microfluidic systems, specific single cells can be selected from a suspension. The selected cells can be sorted, transferred into other mediums without the use of centrifugation and brought into contact with each other in a controlled manner for fusion or signal transfer purposes. This is of interest, for example, for immunological cell activation or the initiation of stem cell differentiation. Binding forces between two cells or micro-scale objects can also be quantified using our technology.

Electrofusion of a P3X myeloma cell and a B cell blast in a dielectric field cage

The cells are first trapped by dielectrophoretic forces and brought into close membrane contact. A short high voltage/low frequency AC pulse (indicated by a flash light) initiates cell fusion. Approx. 2 min after pulse application, the cell membranes at the contact area start to vanish and become more and more fuzzy during the next minute. Approx. 3 min after the pulse application, they have completely disappeared and a single heterokaryon has formed.

Contact-free single-cell handling in a dielectrophoresis-based microfluidic system

By means of consecutive deflection and switch electrodes (black, active electrodes are indicated by a white line), individual cells are selected from their respective flows (arrows), transferred to another flow regime and finally directed to a zigzag electrode. Here, they are held against the fluid flow and are now ready to be flushed with active compounds.

Cell rotation of a mammalian cell in a dielectrophoresis-based microfluidic system

Initially, the cell is dielectrophoretically trapped in a dielectric field cage comprising eight electrodes (black), four of which are located below the cell (i.e., on the bottom of the microchannel) and four of which are located above the cell (i.e., on the top of the microchannel). Selecting phase shifts of 90° between external radio-frequency signals applied to these eight electrodes controls the direction of rotation of the trapped cell around any axis of choice. Scale bar, 10 µm.

Pair formation of a single T cell with a bioactive microbead employing a dielectrophoresis-based microfluidic system

Individual particles of both species are dielectrophoretically selected from their respective particle flows to a zigzag element which retains the objects against the fluid flow going from left to right. Directing both the T lymphocytes and the microbeads to the same zigzag element results ultimately in the formation of stable cell/bead pairs.

Microfluidic test stations for hemo-compatibility tests

Microfluidic system for the precise monitoring of the cellular micro-environment. Using HPLC hoses, the system can be connected to precision syringe pumps and is accessible to high-definition microscopy.
© Photo Fraunhofer IZI

Microfluidic system for the precise monitoring of the cellular micro-environment. Using HPLC hoses, the system can be connected to precision syringe pumps and is accessible to high-definition microscopy.

 

Cell culture-based test systems are an important diagnostic tool in the course of developing bio-tolerated implant surfaces for evaluating the interaction of new materials with living tissue without a great investment in time and expense. In the case of cardiovascular implants, flow-dependent reactions of the coagulation system pose special challenges on the test environment, since parameters such as sample geometry, flow rates and flow conditions must be taken into account.

The objective of the current project is to develop micro-fluidic test benches for efficiently evaluating the hemo-compatibility of coatings of cardiovascular implants as well as the hemo-compatibility of entire implant components, parallelised and in vitro under controlled test conditions (e.g. flow, media composition etc...) as well as at lower material costs than previously possible.

 

Microparticles as sensors for bioanalytics

Accumulation of microparticles in a microchannel using dielectrophoresis. The particles are held back against the fluid flow (red arrows), contact free, and can be released again at the touch of a button.
© Photo Fraunhofer IZI

Accumulation of microparticles in a microchannel using dielectrophoresis. The particles are held back against the fluid flow (red arrows), contact free, and can be released again at the touch of a button.

The majority of bioanalytical procedures still require components and equipment which are too elaborate and expensive. As part of this project, dielectric microparticles will be developed as sensors for biomolecules based on so-called "whispering gallery modes" (WGM) for use in microfluidic components. The advantages of these particles are perfectly showcased here: they can be applied very flexibly as they are able to diffuse freely in the analyte; they require the very smallest sample volumes and can be selected using the simplest methods.

Microsystems for monitoring neuronal cell growth

Neuronal network on a cell culture substrate with a micropatterned coating comprising thermoresponsive polymers. The position of the cell bodies is predefined by the geometric structure of the surface coating. The neurites, through which the individual groups of cells are connected with one another, can be clearly recognized.
© Photo Fraunhofer IZI

Neuronal network on a cell culture substrate with a micropatterned coating comprising thermoresponsive polymers. The position of the cell bodies is predefined by the geometric structure of the surface coating. The neurites, through which the individual groups of cells are connected with one another, can be clearly recognized.

Analysing artificial neuronal networks is a promising way of addressing numerous neurobiological issues. Despite intensive efforts around the world, however, no satisfactory solution has yet been discovered for monitoring the synaptic transfer direction in vitro between the single cells within this type of network, making it difficult to clarify the relationship between form and function in neuronal tissue.

Using micro-manufacturing technologies and in close cooperation with the adjacent Microsystems for In Vitro Cell Models unit, cell culture substrates with surface coatings made from thermoresponsive polymers (TRPs) will be developed in this project, which can then be used to generate neuronal networks with a defined connection pattern. Depending on the temperature, the TRPs applied for this purpose can be transferred from a cell-repellent to a cell-adhesive state, allowing the accessibility of a TRP-coated substrate surface to be controlled to the micrometer for cells and growing neurites. The aim here is to discover new methodical approaches to important neuroscientific issues in foundational research or within the context of pharmaceutical drug development.

A photonic-microfluidic production process for the ultrafast production of customized monoclonal antibodies

Cell pairs in the microfluidic system. The time-lapse sequence shows the highly controlled fusion process of the cells. Fusion is triggered by an electrical impulse to the micro-electrodes surrounding the cell pair (shown in black in the image). Metering bar: 10 µm. Time between the images: 1 min.
© Photo Fraunhofer IZI

Cell pairs in the microfluidic system. The time-lapse sequence shows the highly controlled fusion process of the cells. Fusion is triggered by an electrical impulse to the micro-electrodes surrounding the cell pair (shown in black in the image). Metering bar: 10 µm. Time between the images: 1 min.

Logo EU

Monoclonal antibodies are some of the most frequently used binding molecules worldwide. The core component of the conventional production process is the (uncontrolled) fusion of myeloma cells and B-cells. In this case, individual cells that produce antibodies with the desired bonding properties must be identified from millions of undesired bi-products using elaborate selection steps. This drives up the effort and cost of developing antibodies enormously.

The aim of the project is to establish a procedure in which suitable B-cells can be identified fluorescently before fusion and in which they are subjected to controlled fusion with myeloma cells at single-cell level using a microfluidic protocol. This makes the elaborate post-fusion selection steps superfluous, which reduces effort and costs to a minimum, thereby enabling the production of customized antibodies in less than three weeks.

The project is being carried out in close cooperation with the Chair of Physical Chemistry and the Immune Technologies Working Group at Potsdam University. It receives financial support from the European Union.

  • Microfluidics with computer-controlled pump systems
  • Confocal laser scanning microscope (Zeiss LSM 510)
  • Fully automated fluorescence microscopes for time-lapse recording of living cells (Olympus Cell^R)
  • Laser tweezers / optical tweezers with laser micro-dissection (Palm/Zeiss)
  • Microcontact printer (GeSiM)
  • Dielectrophoresis with computed-controlled 32-channel generators for single cell manipulation and handling of small particle counts in microfluidic chips.
  • Excimer laser ablation unit (wavelength: 248 nm)
  • Transmitted-light and reflected-light microscopy with brightfield, phase contrast, fluorescence, polarization and total internal reflection mode (TIRFM) besides computer-controlled and tempered object plates and time-lapse facility
  • Imaging infrared thermometry
  • CAD design facility
  • Confocal scanning laser microscope with 3D image processing
  • Numerical calculations using the finite element method
  • Optical tweezers (laser tweezers) with combined UV laser for laser cutting
  • Freezing point osmometry
  • Photolithographic process line
  • Micromechanical workshop
  • Bloodwork laboratory

  • Fraunhofer Institute for Applied Polymer Research, research division Life Science and Bioprocesses
  • GeSiM Gesellschaft fuer Silizium-Mikrosysteme mbH
  • Surflay Nanotec GmbH
  • NanoBioAnalytics
  • University of Rostock, Chair of Biophysics
  • Tel Aviv University, OMNI Group
  • microfluidic ChipShop GmbH

Publications

  • Habaza M, Kirschbaum M, Guernth-Marschner C, Dardikman G, Barnea I, Korenstein R, Duschl C, Shaked NT. Rapid 3D Refractive-Index Imaging of Live Cells in Suspension without Labeling Using Dielectrophoretic Cell Rotation. Advanced Science (2016), in press
  • Kirschbaum M, Jaeger MS, Duschl C. Measurement of surface-mediated Ca2+ transients on the single-cell level in a microfluidic Lab-on-a-Chip environment. Methods Mol Biol. (2015);1272:247-56
  • Schreml S, Meier RJ, Kirschbaum M et al. Luminescent Dual Sensors Reveal Extracellular pH-Gradients and Hypoxia on Chronic Wounds That Disrupt Epidermal Repair. Theranostics. (2014), 4, S. 721-735.
  • Guernth-Marschner C, Kirschbaum M, Jaeger MS, Duschl C. Electrofusion of cells in microdevices. Cell News. (2013), 39(3), 14-18.
  • Kirschbaum M, Gürnth-Marschner CR, Cherré S, de Pablo Peña A, Jäger MS, Kroczek RA, Schnelle T, Müller T, Duschl C. Highly controlled single-cell electrofusion in dielectrophoretic field cages. Lab on a Chip. (2012), 12, S. 443-450.
  • Guido I, Xiong C, Jaeger MS, Duschl C. Microfluidic system for cell mechanics analysis through dielectrophoresis. Microelectron Eng. (2012), 97:379-382
  • Boettcher M, Schmidt S, Latz A, Jaeger MS, Stuke M, Duschl C. Filtration at the microfluidic level: enrichment of nanoparticles by tunable filters. J Phys Condens Mat. (2011), 23, 324101
  • Guido I, Jaeger MS, Duschl C. Dielectrophoretic stretching of cells allows for characterization of their mechanical properties. Eur Biophys J. (2011), 40:281-288.
  • Guido I, Jaeger MS, Duschl C. Influence of medium consumption on cell elasticity. Cytotechnology. (2010), 62, 257-263.
  • Kirschbaum M, Jaeger MS, Duschl C. Correlating short-term Ca2+ responses with long-term protein expression after activation of single T cells. Lab Chip. (2009), 9, 3517-3525.
  • Kirschbaum M, Jaeger MS, Schenkel T, Breinig T, Meyerhans A, Duschl C. T cell activation on a single-cell level in dielectrophoresis-based microfluidic devices. J Chromatogr A. (2008), 1202, 83–89.
  • Böttcher M, Jäger MS, Kirschbaum M, Müller T, Schnelle T, Duschl C. Gravitation-driven stress-reduced cell handling. Anal Bioanal Chem. (2008), 390, 857-863.
  • Storn V, Kirschbaum M, Schlosshauer B, Mack AF, Fricke C. Electrical stimulation-induced release of beta-endorphin from genetically modified neuro-2a cells. Cell Transplant. (2008), 17(5):543-8
  • Fiedler S, Müller T, Zwanzig M, Jäger MS, Böttcher M, Csáki A, Fritzsche W, Howitz S, Schmitt D, Hampp N, Scheel W, Fuhr GR, Reichl H. Touchless Component Handling Towards Converging Assembly Strategies. mst news. (2008), 3, 25-28.
  • Jaeger MS, Uhlig K, Schnelle T, Mueller T. Contact-free single-cell cultivation by negative dielectrophoresis. J Phys D Appl Phys. (2008), 41:175502.
  • Jaeger MS, Mueller T, Schnelle T. Thermometry in dielectrophoresis chips for contact-free cell handling. J Phys D Appl Phys. (2007), 40:95–105
  • Böttcher M, Jäger MS, Riegger L, Ducrée J, Zengerle R, Duschl C. Lab-on-chip-based cell separation by combining dielectrophoresis and centrifugation. BRL. (2006), 1(4):443-451.
  • Wiklund M, Guenther C, Lemor R, Jaeger M, Fuhr G, Hertz HM. Ultrasonic standing wave manipulation technology integrated into a dielectrophoretic chip. Lab Chip. (2006), 6:1537–1544.
  • Stuke M, Mueller K, Mueller T, Hagedorn R, Jaeger MS, Fuhr GR. Laser-direct-write creation of three-dimensional OREST microcages for contact-free trapping, handling and transfer of small polarizable neutral objects in solution. Appl Phys A. (2005), 81:915-22.