Cell Analytics

One difficulty in assessing toxicity measurements of new substances such as pharmaceuticals, cosmetics or chemicals/REACH (European Chemicals Regulation on Registration, Evaluation, Authorization and Restriction of Chemicals) is the investigation of meaningful measurands that provide information on the state of the cell tissue. Currently, mainly endpoint analyses (cell staining, gene, protein expression) are performed, which have to be individually adapted to the respective questions. A more generally applicable alternative is based on the measurement of metabolic processes of the cell tissue. By integrating sensors into microfluidic systems for real-time analysis of cell viability, changes in cell metabolism are brought into the temporal context of changes in cultivation conditions, e.g., drug addition and removal. This allows a much more comprehensive elucidation of the mechanisms of action of specific groups of substances and the investigation of repeated doses and combination treatments. For example, cell respiration and pH changes in the immediate vicinity of the cells as well as glucose and lactate concentrations in the cell medium are recorded.

Chemotaxis refers to the directed migration of cells in the concentration gradient of a stimulating substance and plays a central role in many important biological processes such as embryonic development, immune defense or cancer. Understanding the underlying molecular mechanisms is essential for developing new drugs targeting these processes. Unfortunately, classical chemotaxis assays often fail to adequately control the experimental framework for studying chemotaxis. To overcome these limitations, we use microfluidic systems that can generate highly stable concentration gradients and dynamically record the migration of stimulated cells over hours. Here, the gradients are generated under laminar flow conditions by diffusion between two parallel partial flows. By varying flow rates, the steepness of the gradient can be adjusted and it can be positioned at different positions in the microfluidic channel. This allows the stimulation of cells with soluble factors to be varied very flexibly in terms of location and time and adapted to the respective experimental conditions.

The interaction of cells with their environment is a powerful mechanism to control cellular states in vivo. To decipher the signal transduction processes involved in this process, specific events along the cellular signaling cascade need to be detected and their interrelationships elucidated. This cannot be done by ensemble measurements, since averaging biological data always disregards the variability of the response behavior of individual cells and thus provides blurred results. Only multiparameter analysis at the single cell level can provide the crucial information that is essential for a detailed understanding of cellular signaling pathways. In our group, we are developing techniques and procedures to specifically manipulate the microenvironment of individual cells, allowing soluble or surface-bound stimuli to be presented to the cells individually and in a highly controlled manner. The cellular signal transduction processes induced by these stimuli are analyzed at different temporal scales. In this way, important biological processes such as intercellular communication or the differentiation of cells can be studied very precisely and under reproducible environmental conditions.

As we know today, the differentiation of stem cells in the organism is strongly controlled by their microenvironment. Other cell types as well as extracellular proteins play a central role in this process. Via changes in this so-called "niche", the stem cell is given a signal, e.g. to rest, to proliferate or to differentiate.

Unfortunately, observing such dependencies is technically difficult, since one does not work with a flat layer of cells in culture, but in the nascent embryo or in the adult organism one has to try to follow, microscope, stain and manipulate individual cells without exerting an undesirable influence on them. This is sometimes done on simple organisms such as C. elegans (nematode) and D. melanogaster (fruit fly). However, on more complex systems, especially in humans, one faces insurmountable difficulties.

We are developing approaches to artificially recreate the "niche" of stem cells and thus export them under a microscope. This will make it possible to study the behavior of individual stem cells in their physiological environment and to gain valuable insights into basic principles such as ways to influence stem cell differentiation. A distant goal is the controlled differentiation and preparation of single cells in chip format.

• Establishment of functional cell assays (e.g. proliferation, cytotoxicity, chemotaxis, neurite growth, stem cell differentiation, intracellular calcium, intracellular pH, etc.).
• Optical microscopy at high end level (e.g. high light sensitive fluorescence measurements, time resolved fluorescence microscopy of living cell systems, etc...)
• Expression analysis by immunostaining and Western blots
• Detection and quantification of cellular and proteingenic blood components
• Confocal laser scanning microscope (Zeiss LSM510)
• Fully automated fluorescence microscopes with climate control for long-term observation of living cells (cellSens, Olympus)
• Transmitted and reflected light microscopy with bright field, phase contrast, fluorescence, polarization and total internal reflection (TIRFM)
• Systems for real-time measurement of oxygen consumption (Opal, Colibri) and pH changes near cells

• Flechner M, Schaller J, Stahl M, Achberger K, Gerike S, Hannappel Y, Fu J, Jaeger M, Hellweg T, Duschl C, Uhlig K. Adhesion, proliferation and detachment of various cell types on thermoresponsive microgel coatings. Biotechnol Bioeng. (2022), 1– 12.
• Gehre C, Flechner M, Kammerer S, Küpper J-H, Coleman C D, Püschel G P, Uhlig K, Duschl C. Real time monitoring of oxygen uptake of hepatocytes in a microreactor using optical microsensors. Sci Rep (2020) 10, 13700.
• 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
• 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.
• 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.
• 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. Adv. Sci. (2017), 4, 1600205
• 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.
• 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.
• 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.

• Tel Aviv University, OMNI Group
• Universitätsklinikum Regensburg
• Universitätsklinikum Tübingen
• Surflay Nanotec GmbH
• Colibri Photonics GmbH