Interaction of Lysozyme with Poly(L-lysine)/Hyaluronic Acid Multilayers: An ATR-FTIR Study.

Polyelectrolyte multilayers (PEM) loaded with bioactive molecules such as proteins serve as excellent mimics of an extracellular matrix and may find applications in fields such as biomedicine and cell biology. A question which is crucial to the successful employment of PEMs is whether conformation and bioactivity of the loaded proteins is preserved. In this work, the polarized attenuated total reflection Fourier transform infrared (ATR-FTIR) technique is applied to investigate the conformation of the protein lysozyme (Lys) loaded into the poly(L-lysine)/hyaluronic acid (PLL/HA) multilayers.

Adhesion, proliferation, and detachment of various cell types on thermoresponsive microgel coatings.

Cutting-edge biomedical applications require increasingly complex and fastidious cell systems, for example, various classes of primary or stem cells. Their cultivation, however, still differs little from 30 years ago. This especially applies to the use of indiscriminative proteases for nonspecific cell detachment.

Real time monitoring of oxygen uptake of hepatocytes in a microreactor using optical microsensors.

Most in vitro test systems for the assessment of toxicity are based on endpoint measurements and cannot contribute much to the establishment of mechanistic models, which are crucially important for further progress in this field. Hence, in recent years, much effort has been put into the development of methods that generate kinetic data. Real time measurements of the metabolic activity of cells based on the use of oxygen sensitive microsensor beads have been shown to provide access to the mode of action of compounds in hepatocytes.

Label-free discrimination and selection of cancer cells from blood during flow using holography-induced dielectrophoresis.

We present a method for label-free imaging and sorting of cancer cells in blood, which is based on a dielectrophoretic microfluidic chip and label-free interferometric phase microscopy. The chip used for imaging has been embedded with dielectrophoretic electrodes, and therefore it can be used to sort the cells based on the decisions obtained during the cell flow by the label-free quantitative imaging method. Hence, we obtained a real-time, automatic, label-free imaging flow cytometry with the ability to sort the cells during flow.

Combining dielectrophoresis and computer vision for precise and fully automated single-cell handling and analysis.

With the advent of single-cell technologies comes the necessity for efficient protocols to process single cells. We combine dielectrophoresis with open source computer vision programming to automatically control the trajectories of single cells inside a microfluidic device. Using real-time image analysis, individual cells are automatically selected, isolated and spatially arranged.

Micropatterned Thermoresponsive Cell Culture Substrates for Dynamically Controlling Neurite Outgrowth and Neuronal Connectivity in Vitro.

In vitro cultured neuronal networks with defined connectivity are required to improve neuronal cell culture models. However, most protocols for their formation do not provide sufficient control of the direction and timing of neurite outgrowth with simultaneous access for analytical tools such as immunocytochemistry or patch-clamp recordings. Here, we present a proof-of-concept for the dynamic (i.

Thermoresponsive Microgel Coatings as Versatile Functional Compounds for Novel Cell Manipulation Tools.

For the effective use of live cells in biomedicine as in vitro test systems or in biotechnology, non-invasive cell processing and characterisation are key elements. Thermoresponsive polymer coatings have been demonstrated to be highly beneficial for controlling the interaction of adherent cells through their cultivation support. However, the widespread application of these coatings is hampered by limitations in their adaptability to different cell types and because the full range of applications has not yet been fully explored.

Multi-Fractional Analysis of Molecular Diffusion in Polymer Multilayers by FRAP: A New Simulation-Based Approach.

Comprehensive analysis of the multifractional molecular diffusion provides a deeper understanding of the diffusion phenomenon in the fields of material science, molecular and cell biology, advanced biomaterials, etc. Fluorescence recovery after photobleaching (FRAP) is commonly employed to probe the molecular diffusion. Despite FRAP being a very popular method, it is not easy to assess multifractional molecular diffusion due to limited possibilities of approaches for analysis.

A "Cell-Friendly" Window for the Interaction of Cells with Hyaluronic Acid/Poly-l-Lysine Multilayers.

Polyelectrolyte multilayers assembled from hyaluronic acid (HA) and poly-l-lysine (PLL) are most widely studied showing excellent reservoir characteristics to host molecules of diverse nature; however, thick (HA/PLL) films are often found cell repellent. By a systematic study of the adhesion and proliferation of various cells as a function of bilayer number "n" a correlation with the mechanical and chemical properties of films is developed. The following cell lines have been studied: mouse 3T3 and L929 fibroblasts, human foreskin primary fibroblasts VH-Fib, human embryonic kidney HEK-293, human bone cell line U-2-OS, Chinese hamster ovary CHO-K and mouse embryonic stem cells.

Rapid 3D Refractive-Index Imaging of Live Cells in Suspension without Labeling Using Dielectrophoretic Cell Rotation.

A major challenge in the field of optical imaging of live cells is achieving rapid, 3D, and noninvasive imaging of isolated cells without labeling. If successful, many clinical procedures involving analysis and sorting of cells drawn from body fluids, including blood, can be significantly improved. A new label-free tomographic interferometry approach is presented.

Biodegradation-Resistant Multilayers Coated with Gold Nanoparticles. Toward a Tailor-made Artificial Extracellular Matrix.

Polymer multicomponent coatings such as multilayers mimic an extracellular matrix (ECM) that attracts significant attention for the use of the multilayers as functional supports for advanced cell culture and tissue engineering. Herein, biodegradation and molecular transport in hyaluronan/polylysine multilayers coated with gold nanoparticles were described. Nanoparticle coating acts as a semipermeable barrier that governs molecular transport into/from the multilayers and makes them biodegradation-resistant.

Mobility of lysozyme in poly(l-lysine)/hyaluronic acid multilayer films.

The spatial and temporal control over presentation of protein-based biomolecules such as growth factors and hormones is crucial for in vitro applications to mimic the complex in vivo environment. We investigated the interaction of a model protein lysozyme (Lys) with poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films. We focused on Lys diffusion as well as adsorption and retention within the film as a function of the film deposition conditions and post-treatment.

Temperature effect on the build-up of exponentially growing polyelectrolyte multilayers. An exponential-to-linear transition point.

In this study, the effect of temperature on the build-up of exponentially growing polyelectrolyte multilayer films was investigated. It aims at understanding the multilayer growth mechanism as crucially important for the fabrication of tailor-made multilayer films. Model poly(L-lysine)/hyaluronic acid (PLL/HA) multilayers were assembled in the temperature range of 25-85 °C by layer-by-layer deposition using a dipping method.

Patterned Thermoresponsive Microgel Coatings for Noninvasive Processing of Adherent Cells.

Cultivation of adherently growing cells in artificial environments is of utmost importance in medicine and biotechnology to accomplish in vitro drug screening or to investigate disease mechanisms. Precise cell manipulation, like localized control over adhesion, is required to expand cells, to establish cell models for novel therapies and to perform noninvasive cell experiments. To this end, we developed a method of gentle, local lift-off of mammalian cells using polymer surfaces, which are reversibly and repeatedly switchable between a cell-attractive and a cell-repellent state.

Real-time monitoring of oxygen uptake in hepatic bioreactor shows CYP450-independent mitochondrial toxicity of acetaminophen and amiodarone.

Prediction of drug-induced toxicity is complicated by the failure of animal models to extrapolate human response, especially during assessment of repeated dose toxicity for cosmetic or chronic drug treatments. In this work, we present a 3D microreactor capable of maintaining metabolically active HepG2/C3A spheroids for over 28 days in vitro under stable oxygen gradients mimicking the in vivo microenvironment. Mitochondrial respiration was monitored using two-frequency phase modulation of phosphorescent microprobes embedded in the tissue.

Hyaluronic Acid/Poly-l-Lysine Multilayers as Reservoirs for Storage and Release of Small Charged Molecules.

Polyelectrolyte multilayer films are nowadays very attractive for bioapplications due to their tunable properties and ability to control cellular response. Here we demonstrate that multilayers made of hyaluronic acid and poly-l-lysine act as high-capacity reservoirs for small charged molecules. Strong accumulation within the film is explained by electrostatically driven binding to free charges of polyelectrolytes.

Measurement of surface-mediated Ca2+ transients on the single-cell level in a microfluidic lab-on-a-chip environment.

Understanding the dynamics of signal transduction processes that are induced by cell-cell or cell-surface interactions requires the physical stimulation of the cells of interest on a single-cell level and without any ill-defined contacting of their cell membrane. However, standard cell culture techniques are inapplicable for this task as they do not provide cell and particle handling at sufficiently high spatial and temporal resolution and are limited to ensemble measurements. Here, we present a novel process line for the individual stimulation of single cells with bioactive surfaces, like other cells or particles, and the simultaneous analysis of the induced cytosolic calcium signals.

Microporous polymeric 3D scaffolds templated by the layer-by-layer self-assembly.

Polymeric scaffolds serve as valuable supports for biological cells since they offer essential features for guiding cellular organization and tissue development. The main challenges for scaffold fabrication are i) to tune an internal structure and ii) to load bio-molecules such as growth factors and control their local concentration and distribution. Here, a new approach for the design of hollow polymeric scaffolds using porous CaCO3 particles (cores) as templates is presented.

Long-term microfluidic glucose and lactate monitoring in hepatic cell culture.

Monitoring cellular bioenergetic pathways provides the basis for a detailed understanding of the physiological state of a cell culture. Therefore, it is widely used as a tool amongst others in the field of in vitro toxicology. The resulting metabolic information allows for performing in vitro toxicology assays for assessing drug-induced toxicity.

Luminescent dual sensors reveal extracellular pH-gradients and hypoxia on chronic wounds that disrupt epidermal repair.

Wound repair is a quiescent mechanism to restore barriers in multicellular organisms upon injury. In chronic wounds, however, this program prematurely stalls. It is known that patterns of extracellular signals within the wound fluid are crucial to healing.

On the influence of the architecture of poly(ethylene glycol)-based thermoresponsive polymers on cell adhesion.

Thermoresponsive polymer surface coatings are a promising tool for cell culture applications. They allow for a mild way of cell detachment that preserves the activity of membrane proteins-a prerequisite for reliable cell analysis. To enlarge the application range of these coatings to cells with different adhesion properties, we synthesized various novel poly(ethylene glycol)-based thermoresponsive polymers and describe how (i) their chemical structure and (ii) their surface density affect their efficiency.

Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system.

Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates.

Microfluidics as a tool to understand the build-up mechanism of exponential-like growing films.

Microfluidics is used here for the first time to efficiently tune the growth conditions for understanding the build-up mechanism of exponentially growing polyelectrolyte (PE) films. The velocity of PE supply and time of interaction can be successfully altered during the layer-by-layer assembly. Another advantage of this method is that the deposition of poly-L-lysine/hyaluronic acid (PLL/HA) films in microchannels can be monitored online by fluorescence microscopy.

Control of cell adhesion by mechanical reinforcement of soft polyelectrolyte films with nanoparticles.

Chemical cross-linking is the standard approach to tune the mechanical properties of polymer coatings for cell culture applications. Here we show that the elastic modulus of highly swollen polyelectrolyte films composed of poly(L-lysine) (PLL) and hyaluronic acid (HA) can be changed by more than 1 order of magnitude by addition of gold nanoparticles (AuNPs) in a one-step procedure. This hydrogel-nanoparticle architecture has great potential as a platform for advanced cell engineering application, for example remote release of drugs.

Microfluidics meets soft layer-by-layer films: selective cell growth in 3D polymer architectures.

We present here the micropatterns of layer-by-layer (LbL) assembled soft films generated using microfluidic platform that can be exploited for selective cell growth. Using this method, the issue of cell adhesion and spreading on soft LbL-derived films, and simultaneous utilisation of such unmodified soft films to exploit their reservoir properties are addressed. This also paves the way for extending the culture of cells to soft films and other demanding applications like triggered release of biomolecules.