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. Spectra are taken from the protein in the PEMs coated onto an ATR crystal during protein adsorption and desorption. For comparison, a similar investigation is performed for the case of Lys in contact with the uncoated crystal. The study highlights the presence of both "tightly" and "poorly bound" Lys fractions in the PEM. These fractions differ in their conformation and release behavior from the PEM upon washing. Comparison of spectra recorded with different polarizations suggests preferential orientation of alpha helical structures, beta sheets and turns in the "tightly bound" Lys. In contrast, the "poorly bound" fraction shows isotropic orientation and its conformation is well preserved.

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. A far more gentle alternative changes the adhesive properties of the cell culture substrates through coatings based on thermoresponsive polymers. Such polymers mediate cell adhesion at 37°C, but become repulsive upon a cell-compatible temperature drop to, for example, 32°C. While the high functionality of this method has already been well proven, it must also be easy and reproducible to apply. Here, we emphasize the potential of standard cell culture materials coated by spraying with thermoresponsive microgels for routine cultivation and beyond. On these surfaces, we successfully cultivated and detached various cell types, including induced pluripotent stem cells and cells in serum-free culture. In addition, we evaluated the compatibility of the microgel-sprayed surfaces with adhesion-promoting proteins, which are essential for, for example, stem cells or neuronal cells. Finally, we demonstrate that the microgel surfaces do not impair proliferation and show their long-term stability. We conclude that for cell detachment, thermoresponsive cell culture substrates can fully substitute proteases, like trypsin, by employing a comparably straightforward protocol that is compatible with many industrial processing lines.

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. However, for fully exploiting this approach a detailed knowledge of the microenvironment of the cells is required. In this work, we investigate the cellular behaviour of three types of hepatocytes, HepG2 cells, HepG2-3A4 cells and primary mouse hepatocytes, towards their exposure to acetaminophen when the availability of oxygen for the cell is systematically varied. We show that the relative emergence of two modes of action, one NAPQI dependent and the other one transient and NAPQI independent, scale with expression level of CYP3A4. The transient cellular response associated to mitochondrial respiration is used to characterise the influence of the initial oxygen concentration in the wells before exposure to acetaminophen on the cell behaviour. A simple model is presented to describe the behaviour of the cells in this scenario. It demonstrates the level of control over the role of oxygen supply in these experiments. This is crucial for establishing this approach into a reliable and powerful method for the assessment of toxicity.

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. To validate our model, we combined into the label-free imaging interferometer a fluorescence imaging channel that indicated the correctness of the label-free sorting. We have achieved above 98% classification success and 69% sorting accuracy at flow rates of 4 to 7 µL hr-1 . In the future, this method is expected to help in label-free sorting of circulating tumor cells in blood following an initial state-of-the-art cell enrichment.

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.e., time-gated) control of neurite outgrowth on a cell culture substrate based on 2D-micropatterned coatings of thermoresponsive polymers (TRP). The pattern consists of uncoated microstructures where neurons can readily adhere and neurites can extend along defined pathways. The surrounding regions are coated with TRP that does not facilitate cell or neurite growth at 33 °C. Increasing the ambient temperature to 37 °C renders the TRP coating cell adhesive and enables the crossing of gaps coated with TRP by neurites to contact neighboring cells. Here, we demonstrate the realization of this approach employing human neuronal SH-SY5Y cells and human induced neuronal cells. Our results suggest that this approach may help to establish a spatiotemporal control over the connectivity of multinodal neuronal networks.

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. In the work presented here, we address these issues by focusing on three different aspects. With regard to the first aspect, by using well-defined laminar flow in a microchannel, a highly controllable and reproducible shear force can be applied to adherent cells. Employing this tool, we demonstrate that cells can be non-invasively detached from a support using a defined shear flow. The second aspect relates to the recent development of simple methods for patterning thermoresponsive coatings. Here, we show how such patterned coatings can be used for improving the handling and reliability of a wound-healing assay. Two pattern geometries are tested using mouse fibroblasts and CHO cells. In terms of the third aspect, the adhesiveness of cells depends on the cell type. Standard thermoresponsive coatings are not functional for all types of cells. By coadsorbing charged nanoparticles and thermoresponsive microgels, it is demonstrated that the adhesion and detachment behaviour of cells on such coatings can be modulated.

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. Here we present a novel simulation-optimization-based approach (S-approach) that significantly broadens possibilities of the analysis. In the S-approach, possible fluorescence recovery scenarios are primarily simulated and afterward compared with a real measurement while optimizing parameters of a model until a sufficient match is achieved. This makes it possible to reveal multifractional molecular diffusion. Fluorescent latex particles of different size and fluorescein isothiocyanate in an aqueous medium were utilized as test systems. Finally, the S-approach has been used to evaluate diffusion of cytochrome c loaded into multilayers made of hyaluronan and polylysine. Software for evaluation of multifractional molecular diffusion by S-approach has been developed aiming to offer maximal versatility and user-friendly way 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)n 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. All cells adhere and spread well in a narrow "cell-friendly" window identify in the range of n = 12-15. At n < 12, the film is inhomogeneous and at n > 15, the film is cell repellent for all cell lines. Cellular adhesion correlates with the mechanical properties of the films showing that softer films at higher "n" number exhibiting a significant decrease of the Young's modulus below 100 kPa are weakly adherent to cells. This trend cannot be reversed even by coating a strong cell-adhesive protein fibronectin onto the film. This indicates that mechanical cues plays a major role for cell behavior, also in respect to biochemical ones.

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. This approach provides rapid capturing of the 3D refractive-index distribution of single cells in suspension. The cells flow in a microfluidic channel, are trapped, and then rapidly rotated by dielectrophoretic forces in a noninvasive and precise manner. Interferometric projections of the rotated cell are acquired and processed into the cellular 3D refractive-index map. Uniquely, this approach provides full (360°) coverage of the rotation angular range around any axis, and knowledge on the viewing angle. The experimental demonstrations presented include 3D, label-free imaging of cancer cells and three types of white blood cells. This approach is expected to be useful for label-free cell sorting, as well as for detection and monitoring of pathological conditions resulting in cellular morphology changes or occurrence of specific cell types in blood or other body fluids.

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. Model protein lysozyme (mimics of ECM-soluble signals) diffuses into the multilayers as fast- and slow-diffusing populations existing in an equilibrium. Such a composite system may have high potential to be exploited as degradation-resistant drug-delivery platforms suitable for cell-based applications.

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. Additionally, an effect of Lys concentration on its mobility was probed. A combination of confocal fluorescence microscopy, fluorescence recovery after photobleaching, and microfluidics was employed for this investigation. Our main finding is that adsorption of PLL and HA after protein loading induces acceleration and reduction of Lys mobility, respectively. These results suggest that a charge balance in the film to a high extent governs the protein-film interaction. We believe that control over protein mobility is a key to reach the full potential of the PLL/HA films as reservoirs for biomolecules depending on the application demand.

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. The film growth switches from the exponential to the linear regime at the transition point as a result of limited polymer diffusion into the film. With the increase of the build-up temperature the film growth rate is enhanced in both regimes; the position of the transition point shifts to a higher number of deposition steps confirming the diffusion-mediated growth mechanism. Not only the faster polymer diffusion into the film but also more porous/permeable film structure are responsible for faster film growth at higher preparation temperature. The latter mechanism is assumed from analysis of the film growth rate upon switching of the preparation temperature during the film growth. Interestingly, the as-prepared films are equilibrated and remain intact (no swelling or shrinking) during temperature variation in the range of 25-45 °C. The average activation energy for complexation between PLL and HA in the multilayers calculated from the Arrhenius plot has been found to be about 0.3 kJ mol(-1) for monomers of PLL. Finally, the following processes known to be dependent on temperature are discussed with respect to the multilayer growth: (i) polymer diffusion, (ii) polymer conformational changes, and (iii) inter-polymer interactions.

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. This property was introduced through micropatterned thermoresponsive polymer coatings formed from colloidal microgels. Patterning was obtained through automated nanodispensing or microcontact printing, making use of unspecific electrostatic interactions between microgels and substrates. This process is much more robust against ambient conditions than covalent coupling, thus lending itself to up-scaling. As an example, wound healing assays were accomplished at 37 °C with highly increased precision in microfluidic environments.

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. Phase modulation is focus independent and unaffected by cell death or migration. This sensitive measurement of oxygen dynamics revealed important information on the drug mechanism of action and transient subthreshold effects. Specifically, exposure to antiarrhythmic agent, amiodarone, showed that both respiration and the time to onset of mitochondrial damage were dose dependent showing a TC50 of 425 µm. Analysis showed significant induction of both phospholipidosis and microvesicular steatosis during long-term exposure. Importantly, exposure to widely used analgesic, acetaminophen, caused an immediate, reversible, dose-dependent loss of oxygen uptake followed by a slow, irreversible, dose-independent death, with a TC50 of 12.3 mM. Transient loss of mitochondrial respiration was also detected below the threshold of acetaminophen toxicity. The phenomenon was repeated in HeLa cells that lack CYP2E1 and 3A4, and was blocked by preincubation with ascorbate and TMPD. These results mark the importance of tracing toxicity effects over time, suggesting a NAPQI-independent targeting of mitochondrial complex III might be responsible for acetaminophen toxicity in extrahepatic tissues.

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. Binding and release mechanisms are discussed based on charge balance and polymer dynamics in the film. Our results show that transport of molecules through the film-solution interface limits the release rate. The multilayers might serve as an effective platform for drug delivery and tissue engineering due to high potential for drug loading and controlled release.

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. The method is based on a microfluidic lab-on-a-chip environment that allows for contactless cell and particle handling by dielectrophoretic forces.

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. The cores packed into a microfluidic channel are coated with polymers employing the layer-by-layer (LbL) technique. Subsequent core elimination at mild conditions results in formation of the scaffold composed of interconnected hollow polymer microspheres. The size of the cores determines the feature dimensions and, as a consequence, governs cellular adhesion: for 3T3 fibroblasts an optimal microsphere size is 12 µm. By making use of the carrier properties of the porous CaCO3 cores, the microspheres are loaded with BSA as a model protein. The scaffolds developed here may also be well suited for the localized release of bio-molecules using external triggers such as IR-light.

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. Extracellular pH (pHe) is precisely regulated and potentially important in signaling within wounds due to its diverse cellular effects. Additionally, sufficient oxygenation is a prerequisite for cell proliferation and protein synthesis during tissue repair. It was, however, impossible to study these parameters in vivo due to the lack of imaging tools. Here, we present luminescent biocompatible sensor foils for dual imaging of pHe and oxygenation in vivo. To visualize pHe and oxygen, we used time-domain dual lifetime referencing (tdDLR) and luminescence lifetime imaging (LLI), respectively. With these dual sensors, we discovered centripetally increasing pHe-gradients on human chronic wound surfaces. In a therapeutic approach, we identify pHe-gradients as pivotal governors of cell proliferation and migration, and show that these pHe-gradients disrupt epidermal barrier repair, thus wound closure. Parallel oxygen imaging also revealed marked hypoxia, albeit with no correlating oxygen partial pressure (pO2)-gradient. This highlights the distinct role of pHe-gradients in perturbed healing. We also found that pHe-gradients on chronic wounds of humans are predominantly generated via centrifugally increasing pHe-regulatory Na+/H+-exchanger-1 (NHE1)-expression. We show that the modification of pHe on chronic wound surfaces poses a promising strategy to improve healing. The study has broad implications for cell science where spatial pHe-variations play key roles, e.g. in tumor growth. Furthermore, the novel dual sensors presented herein can be used to visualize pHe and oxygenation in various biomedical fields.

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. In this study, we demonstrate the value of a microsystem for the fully automated detection of drug-induced changes in cellular viability by continuous monitoring of the metabolic activity over several days. To this end, glucose consumption and lactate secretion of a hepatic tumor cell line were continuously measured using microfluidically addressed electrochemical sensors. Adapting enzyme-based electrochemical flat-plate sensors, originally designed for human whole-blood samples, to their use with cell culture medium supersedes the common manual and laborious colorimetric assays and off-line operated external measurement systems. The cells were exposed to different concentrations of the mitochondrial inhibitor rotenone and the cellular response was analyzed by detecting changes in the rates of the glucose and lactate metabolism. Thus, the system provides real-time information on drug-induced liver injury in vitro.