Brigitte Angres

Cell adhesion profiling using extracellular matrix protein microarrays.

We have developed a microarray-based system for cell adhesion profiling of large panels of cell-adhesive proteins to increase the throughput of in vitro cell adhesion assays, which are currently primarily performed in multiwell plates. Miniaturizing cell adhesion assays to an array format required the development of protocols for the reproducible microspotting of extracellular matrix (ECM) protein solutions and for the handling of cell suspensions during the assay. We generated ECM protein microarrays with high reproducibility in microspot protein content using nitrocellulose-coated glass microslides, combined with piezoelectric microspotting of protein solutions. Protocols were developed that allowed us to use 5000 cells or fewer on an array of 4 x 4 mm consisting of 64 microspots. Using this microarray system, we identified differences of adhesive properties of three cell lines to 14 different ECM proteins. Furthermore, the sensitivity and accuracy of the assays were increased using microarrays with ranges of ECM protein amounts. This microarray system will be particularly useful for extensive comparative cell adhesion profiling studies when only low amounts of adhesive substrate and cells, such as stem cells or cells from biopsies, are available.

A membrane‐bound FRET‐based caspase sensor for detection of apoptosis using fluorescence lifetime and total internal reflection microscopy

A caspase sensor based on Förster resonance energy transfer between fluorescent proteins is reported. Enhanced cyan fluorescent protein anchored to the inner leaflet of the plasma membrane of living cells is optically excited by an evanescent electromagnetic field and transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein. Upon apoptosis, DEVD is cleaved and energy transfer is disrupted, as proven by pronounced changes in fluorescence spectra and decay times. Fluorescence spectroscopy and lifetime imaging (FLIM) is combined with total internal reflection fluorescence microscopy (TIRFM) for selective detection of this membrane‐bound caspase sensor. Fluorophores of the cytoplasm are thus excluded, and the signal‐to‐background ratio is increased considerably. In comparison with conventional or laser scanning microscopy, this permits long‐term observation of apoptosis in live cell cultures using very low absorption and avoiding light‐induced damages of the samples.

MicroPrep: Chip-based dielectrophoretic purification of mitochondria

We have developed a microfluidic system – microPrep – for subcellular fractionation of cell homogenates based on dielectrophoretic sorting. Separation of mitochondria isolated from a human lymphoblastoid cell line was monitored by fluorescence microscopy and further characterized by western blot analysis. Robust high throughput and continuous long‐term operation for up to 60 h of the microPrep chip system with complex biological samples became feasible as a result of a comprehensive set of technical measures: (i) coating of the inner surfaces of the chip with BSA, (ii) application of mechanical actuators to induce periodic flow patterns, (iii) efficient cooling of the device to ensure integrity of organelle, (iv) a wide channel to provide for high fluidic throughput, and (v) integration of a serial arrangement of 10 dielectrophoretic deflector units to enable separation of samples with a high particle load without clogging. Hence, microPrep yields tens of micrograms of enriched and purified mitochondria within hours. Western blots of mitochondria fractions showed that contaminating endoplasmatic reticulum was reduced by a factor 6 when compared with samples prepared by state of the art centrifugation.

Transforming growth factor β1 and laminin-111 cooperate in the induction of interleukin-16 expression in synovial fibroblasts from patients with rheumatoid arthritis

Objectives: In synovial tissues of patients with rheumatoid arthritis (RA), strong expression of laminins and integrins co-localises with increased expression of inflammatory cytokines. Synovial fibroblasts (SF) contribute to the pathogenesis of RA through increased expression of cytokines and chemoattractant factors, one of which is interleukin-16 (IL16). A study was undertaken to investigate the regulatory pathways of IL16 in SF from patients with RA (RA-SF) and osteoarthritis (OA-SF). Methods: SF were seeded in laminin-coated flasks and activated by the addition of cytokines. The expression of IL16 was investigated by quantitative RT-PCR, immunoblotting and ELISA; its biological activity was determined by a cell migration assay. Cell–matrix interactions were investigated by cell binding and attachment assays. Relevant intracellular signalling pathways were studied by immunoblotting and with pharmacological blocking reagents. Results: Stimulation of SF with transforming growth factor β1 (TGF-β1) and growth on laminin-111 (LM-111) significantly increased the expression of IL16. Binding to LM-111 induced significantly more IL16 mRNA in RA-SF than in OA-SF (p<0.05). The IL16 cytokine was detected in supernatants of TGF-β1-activated and in LM-111+TGF-β1-activated RA-SF (38 to 62 pg/ml), but not in supernatants of OA-SF. This IL16 regulation involved p38MAPK, ERK1/2 and SMAD2 signalling, but not NFκB. Conclusions: Binding of RA-SF to LM-111 in the presence of TGF-β1 triggers a significant IL16 response and thus may contribute to the infiltration of CD4+ lymphocytes into synovial tissues. This mode of IL16 induction represents a novel pathway leading to IL16 production in RA-SF but not in OA-SF, which operates independently of NFκB signalling.

Monitoring of Apoptosis in 3D Cell Cultures by FRET and Light Sheet Fluorescence Microscopy

Non-radiative cell membrane associated Förster Resonance Energy Transfer (FRET) from an enhanced cyan fluorescent protein (ECFP) to an enhanced yellow fluorescent protein (EYFP) is used for detection of apoptosis in 3-dimensional cell cultures. FRET is visualized in multi-cellular tumor spheroids by light sheet based fluorescence microscopy in combination with microspectral analysis and fluorescence lifetime imaging (FLIM). Upon application of staurosporine and to some extent after treatment with phorbol-12-myristate-13-acetate (PMA), a specific activator of protein kinase c, the caspase-3 sensitive peptide linker DEVD is cleaved. This results in a reduction of acceptor (EYFP) fluorescence as well as a prolongation of the fluorescence lifetime of the donor (ECFP). Fluorescence spectra and lifetimes may, therefore, be used for monitoring of apoptosis in a realistic 3-dimensional system, while light sheet based microscopy appears appropriate for 3D imaging at low light exposure.

Numerical modelling and measurement of cell trajectories in 3‐D under the influence of dielectrophoretic and hydrodynamic forces

This research is part of a program aiming at the development of a fluidic microsystem for in vitro drug testing. For this purpose, primary cells need to be assembled to form cellular aggregates in such a way as to resemble the basic functional units of organs. By providing for in vivo‐like cellular contacts, proper extracellular matrix interaction and medium perfusion it is expected that cells will retain their phenotype over prolonged periods of time. In this way, in vitro test systems exhibiting in vivo type predictivity in drug testing are envisioned. Towards this goal a 3‐D microstructure micro‐milled in a cyclic olefin copolymer (COC) was designed in such a way as to assemble liver cells via insulator‐based dielectrophoresis (iDEP) in a sinusoid‐type fashion. First, numeric modelling and simulation of dielectrophoretic and hydrodynamic forces acting on cells in this microsystem was performed. In particular, the problem of the discontinuity of the electric field at the interface between the fluid media in the system and the polymer materials it consists of was addressed. It was shown that in certain cases, the material of the microsystem may be neglected altogether without introducing considerable error into the numerical solution. This simplification enabled the simulation of 3‐D cell trajectories in complex chip geometries. Secondly, the assembly of HepG2 cells by insulator‐based dielectrophoresis in this device is demonstrated. Finally, theoretical results were validated by recording 3‐D cell trajectories and the Clausius–Mossotti factor of liver cells was determined by combining results obtained from both simulation and experiment.

Adhesion to Extracellular Matrix Proteins can Differentiate between Human Bone Marrow Derived Mesenchymal Stem Cells and Fibroblasts

Mesenchymal stem or stromal cells (MSC) contribute in vivo to wound repair and can be utilized for tissue regeneration. In contrast, fibroblasts may contribute to scar formation and may even hamper functional regeneration. Depending on the clinical application, MSC are sometimes attached to a scaffold to maintain the cells in the area of regeneration. We therefore screened for proteins that allow a preferential binding of MSC and avoid strong adherence of fibroblasts. The human MSC were isolated from bone marrow (bmMSC) or term placenta (pMSC). Synovial fibroblasts (SF) and dermal fibroblasts (DF) served as controls. In the first set of experiments, binding of bmMSC and SF to extracellular matrix (ECM) proteins was investigated by multiple substrate array (MSA®). From MSA® protein analyses 57 peptides with potential MSC-binding sites were selected and the binding of the cells to these peptides was determined. We report that MSC differ from fibroblasts in their binding to proteins of the extracellular matrix. MSC bind with higher efficiency to laminin-111, collagens-I, -III, and -IV and tenascin-C compared to fibroblasts, while both cell types bind with high efficiency to fibronectin, vitronectin, and laminin-511. We conclude that overall MSC seem less selective with respect to binding extracellular matrix components compared to fibroblasts, and fibroblasts attach to fewer proteins and peptides.

Förster resonance energy transfer-based total internal reflection fluorescence reader for apoptosis

A fluorescence reader for the detection of Forster resonance energy transfer (FRET) on surfaces of living cells is described. The method is based on multiple total internal reflections (TIR) of an incident laser beam within a glass slide, such that individual samples on top of the glass slide are illuminated simultaneously by an evanescent electromagnetic field. Enhanced cyan fluorescent protein (ECFP) anchored to the inner leaflet of the plasma membrane is optically excited and transfers its excitation energy via the peptide linker Asp-Glu-Val-Asp (DEVD) to an enhanced yellow fluorescent protein. Upon apoptosis, DEVD is cleaved, and energy transfer is disrupted, as proven by an increase of fluorescence intensity as well as of fluorescence lifetime of the donor ECFP. Due to selective excitation of membrane-associated fluorophores, intracellular fluorescence and background luminescence from the surrounding medium are eliminated. Therefore, this test system appears to be a sensitive device for the detection of apoptosis and more generally for drug screening or in vitro diagnosis on a nanometer scale.

Total internal reflection energy transfer (TIRET) microscopy for analysis of focal adhesions in living cells

Total internal reflection fluorescence microscopy (TIRFM) is used to measure non-radiative energy transfer between membrane associated proteins in living cells. Measurements are concentrated on focal contacts and their associated proteins focal adhesion kinase (FAK) and Paxillin (Pax) which play major roles with respect to cell migration, growth, and survival. These proteins are visualized after fusion with variants of green fluorescent protein (ECFP and EYFP), and an intermolecular energy transfer ECFP -> EYFP is deduced from fluorescence spectra as well as from fluorescence decay kinetics of single cells.

Fluorescence intensity, lifetime, and anisotropy screening of living cells based on total internal reflection techniques

A setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of living cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections (TIR) within the bottom of a microtiter plate, such that up to 96 individual samples are illuminated simultaneously by an evanescent electromagnetic field. Two different screening procedures for the detection of fluorescence arising from the plasma membrane of living cells by High Throughput Screening (HTS) and High Content Screening (HCS), are distinguished. In the first case a rapid measurement of large sample numbers based on fluorescence intensity, and in the second case a high content of information from a single sample based on the parameters fluorescence lifetime (Fluorescence Lifetime Screening, FLiS) and fluorescence anisotropy (Fluorescence Lifetime Polarization Screening, FLiPS) is achieved. Both screening systems were validated using cultivated cells incubated with different fluorescent markers (e. g. NBD-cholesterol) as well as stably transfected cells expressing a fluorescent membrane-associating protein. In addition, particularly with regard of potential pharmaceutical applications, the kinetics of the intracellular translocation of a fluorescent protein kinase c fusion protein upon stimulation of the cells was determined. Further, a caspase sensor based on Förster Resonance Energy Transfer (FRET) between fluorescent proteins was tested. Enhanced cyan fluorescent protein (ECFP) anchored to the inner leaflet of the plasma membrane of living cells transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein (EYFP). Upon apoptosis DEVD is cleaved, and energy transfer is disrupted, as proven by changes in fluorescence intensity and decay times.