A high-precision wound healing assay based on photosensitized culture substrates.

Quantitative assessment of cell migration in vitro is often required in fundamental and applied research from different biomedical areas including wound repair, tumor metastasis or developmental biology. A collection of assays has been established throughout the years like the most widely used scratch assay or the so-called barrier assay. It is the principle of these assays to introduce a lesion into an otherwise confluent monolayer in order to study the migration of cells from the periphery into this artificial wound and determine the migration rate from the time necessary for wound closure. A novel assay makes use of photosensitizers doped into a polystyrene matrix. A thin layer of this composite material is coated on the bottom of regular cell culture ware showing perfect biocompatibility. When adherent cells are grown on this coating, resonant excitation of the photosensitizer induces a very local generation of 1O2, which kills the cells residing at the site of illumination. Cells outside the site of illumination are not harmed. When excitation of the photosensitizer is conducted by microscopic illumination, high-precision wounding in any size and geometry is available even in microfluidic channels. Besides proof-of-concept experiments, this study gives further insight into the mechanism of photosensitizer-mediated cell wounding.

Development of Specialized Microelectrode Arrays with Local Electroporation Functionality.

When a cell or tissue is exposed to a pulsed electric field (100-1000 V/cm), the cellular membrane permeabilizes for biomolecules that cannot pass an intact cellular membrane. During this electropermeabilization (EP), plasmid deoxyribonucleic acid sequences encoding therapeutic or regulatory genes can enter the cell, which is called gene electrotransfer (GET). GET using micro-/nano technology provides higher spatial resolution and operates with lower voltage amplitudes compared to conventional bulk EP. Microelectrode arrays (MEAs), which are usually used for the recording and stimulation of neuronal signals, can be utilized for GET as well. In this study, we developed a specialized MEA for local EP of adherent cells. Our manufacturing process provides a most flexible electrode and substrate material selection. We used electrochemical impedance spectroscopy to characterize the impedance of the MEAs and the impact of an adherent cellular layer. We verified the local EP functionality of the MEAs by loading a fluorophore dye into human embryonic kidney 293T cells. Finally, we demonstrated a GET with a subsequent green fluorescent protein expression by the cells. Our experiments prove that a high spatial resolution of GET can be obtained using MEAs.

Monitoring the Reversibility of GPCR Signaling by Combining Photochromic Ligands with Label-free Impedance Analysis.

G protein-coupled cell surface receptors (GPCR) trigger complex intracellular signaling cascades upon agonist binding. Classic pharmacological assays provide information about binding affinities, activation or blockade at different stages of the signaling cascade, but real time dynamics and reversibility of these processes remain often disguised. We show that combining photochromic NPY receptor ligands, which can be toggled in their receptor activation ability by irradiation with light of different wavelengths, with whole cell label-free impedance assays allows observing the cell response to receptor activation and its reversibility over time. The concept demonstrated on NPY receptors may be well applicable to many other GPCRs providing a deeper insight into the time course of intracellular signaling processes.

GPR4 in the pH-dependent migration of melanoma cells in the tumor microenvironment.

Due to its high metastatic potential, malignant melanoma is one of the deadliest skin cancers. In melanoma as well as in other cancers, acidification of the tumor microenvironment (=TME, inverse pH-gradient) is a well-known driver of tumor progression and metastasis. Membrane-bound receptors, such as the proton-sensitive GPCR (pH-GPCR) GPR4, are considered as potential initiators of the signalling cascades relevant to malignant transformation. In this study, we investigated the pH-dependent migration of GPR4 wildtype/overexpressing SK-Mel-28 cells using an impedance-based electrical wounding and migration assay and classical Boyden chamber experiments. Migration of GPR4 overexpressing SK-Mel-28 cells was enhanced in a range of pH 6.5-7.5 as compared to controls in the impedance-based electrical wounding and migration assay. In Boyden chamber experiments, GPR4 overexpression only increased migration at pH 7.5 in a Matrigel-free setup, but not at pH 6.5. Results indicate that GPR4 is involved in the migration of melanoma cells, especially in the tumor periphery, and that this process is affected by pH in the TME.

Label-free impedance measurements to unravel biomolecular interactions involved in G protein-coupled receptor signaling.

G protein-coupled receptors (GPCRs) are among the most heavily addressed drug targets in medicinal chemistry and pharmacology. The screening for new agonists or antagonists has been largely based on genetically engineered cells overexpressing the receptor to study binding of ligands directly or via intracellular signaling events downstream of receptor activation. These approaches are often invasive in nature, need to be conducted as endpoint assays, require isotope- or fluorophore-labeling and significant genetic manipulation. In contrast to that, non-invasive and label-free impedance measurements are capable of monitoring ligand-receptor interactions in target cells with endogenous receptor expression in real time. The cells expressing the receptor are grown on planar gold-film electrodes that are integrated into regular cell culture dishes. This article will highlight several impedance-based assay formats to characterize biomolecular interactions between ligands and their GPCRs in vitro, comprising agonist and antagonist characterization, dose-response relationships, receptor desensitization, and signal transduction profiling.

Innovative Platform for the Advanced Online Monitoring of Three-Dimensional Cells and Tissue Cultures.

The use of 3D cell cultures has gained increasing importance in medical and pharmaceutical research. However, the analysis of the culture medium is hardly representative for the culture conditions within a 3D model which hinders the standardization of 3D cultures and translation of results. Therefore, we developed a modular monitoring platform combining a perfusion bioreactor with an integrated minimally invasive sampling system and implemented sensors that enables the online monitoring of culture parameters and medium compounds within 3D cultures. As a proof-of-concept, primary cells as well as cell lines were cultured on a collagen or gelatin methacryloyl (GelMA) hydrogel matrix, while monitoring relevant culture parameters and analytes. Comparing the interstitial fluid of the 3D models versus the corresponding culture medium, we found considerable differences in the concentrations of several analytes. These results clearly demonstrate that analyses of the culture medium only are not relevant for the development of standardized 3D culture processes. The presented bioreactor with an integrated sampling and sensor platform opens new horizons for the development, optimization, and standardization of 3D cultures. Furthermore, this technology holds the potential to reduce animal studies and improve the transferability of pharmaceutical in vitro studies by gaining more relevant results, bridging the gap towards clinical translation.

Clinical long-term and patient-reported outcomes of dental implants in oral cancer patients.

BACKGROUND AND PURPOSE: The aim of this clinical study was to investigate the clinical long-term and patient-reported outcome of dental implants in patients with oral cancer. In addition, analysis of the influence of radiation therapy, timing of implant insertion, and augmentation procedures on implant survival was performed. MATERIAL AND METHODS: This retrospective study investigated the clinical outcome of 711 dental implants in 164 oral cancer patients, inserted by experienced surgeons of the Department of Oral and Maxillofacial Surgery, University Medical Center Mainz, Germany. Oral health-related quality of life (OHRQoL) was evaluated. RESULTS: Cumulative 5-year and 10-year implant survival rates for all included implants were 87.3% and 80.0%. Implants placed straight after ablative surgery (primary implant placement) and implants placed after completing the oncologic treatment (secondary implant placement) showed a comparable implant survival (92.5% vs. 89.5%; p = 0.635). Irradiation therapy had no significant influence on implant survival of secondary placed implants (p = 0.929). However, regarding implant site (native bone vs. augmented bone) and radiation therapy (non-irradiated bone vs. irradiated bone), implants inserted in irradiated bone that received augmentation procedures showed a statistically significant lower implant survival (p < 0.001). Patients reported a distinct improvement in OHRQoL. CONCLUSIONS: Promising long-term survival rates of dental implants in patients after treatment of oral cancer were seen. In addition, patients benefit in form of an improved OHRQoL. However, bone augmentation procedures in irradiated bone may result in an impaired implants' prognosis.

Impedance analysis of adherent cells after in situ electroporation-mediated delivery of bioactive proteins, DNA and nanoparticles in µL-volumes.

Specific intracellular manipulation of animal cells is a persistent goal in experimental cell biology. Such manipulations allow precise and targeted interference with signaling cascades, metabolic pathways, or bi-molecular interactions for subsequent tracking of functional consequences. However, most biomolecules capable of molecular recognition are membrane impermeable. The ability to introduce these molecules into the cytoplasm and then to apply appropriate readouts to monitor the corresponding cell response could prove to be an important research tool. This study describes such an experimental approach combining in situ electroporation (ISE) as a means to efficiently deliver biomolecules to the cytoplasm with an impedance-based, time-resolved analysis of cell status using electric cell-substrate impedance sensing (ECIS). In this approach, gold-film electrodes, deposited on the bottom of regular culture dishes, are used for both electroporation and monitoring. The design of the electrode layout and measurement chamber allows working with sample volumes as small as 10 µL. A miniaturized setup for combined electroporation and impedance sensing (µISE-ECIS) was applied to load different adherent cells with bioactive macromolecules including enzymes, antibodies, nucleic acids and quantum dot nanoparticles. The cell response after loading the cytoplasm with RNase A or cytochrome c (in the presence or absence of caspase inhibitors) was tracked by non-invasive impedance readings in real-time.

Fasudil Loaded PLGA Microspheres as Potential Intravitreal Depot Formulation for Glaucoma Therapy.

Rho-associated protein kinase (ROCK) inhibitors allow for causative glaucoma therapy. Unfortunately, topically applied ROCK inhibitors suffer from high incidence of hyperemia and low intraocular bioavailability. Therefore, we propose the use of poly (lactide-co-glycolide) (PLGA) microspheres as a depot formulation for intravitreal injection to supply outflow tissues with the ROCK inhibitor fasudil over a prolonged time. Fasudil-loaded microspheres were prepared by double emulsion solvent evaporation technique. The chemical integrity of released fasudil was confirmed by mass spectrometry. The biological activity was measured in cell-based assays using trabecular meshwork cells (TM cells), Schlemm's canal cells (SC cells), fibroblasts and adult retinal pigment epithelium cells (ARPE-19). Cellular response to fasudil after its diffusion through vitreous humor was investigated by electric cell-substrate impedance sensing. Microspheres ranged in size from 3 to 67 µm. The release of fasudil from microspheres was controllable and sustained for up to 45 days. Released fasudil reduced actin stress fibers in TM cells, SC cells and fibroblasts. Decreased collagen gel contraction provoked by fasudil was detected in TM cells (~2.4-fold), SC cells (~1.4-fold) and fibroblasts (~1.3-fold). In addition, fasudil readily diffused through vitreous humor reaching its target compartment and eliciting effects on TM cells. No negative effects on ARPE-19 cells were observed. Since fasudil readily diffuses through the vitreous humor, we suggest that an intravitreal drug depot of ROCK inhibitors could significantly improve current glaucoma therapy particularly for patients with comorbid retinal diseases.

pH sensing in skin tumors: Methods to study the involvement of GPCRs, acid-sensing ion channels and transient receptor potential vanilloid channels.

Solid tumors exhibit an inversed pH gradient with increased intracellular pH (pHi ) and decreased extracellular pH (pHe ). This inside-out pH gradient is generated via sodium/hydrogen antiporter 1, vacuolar-type H + ATPases, monocarboxylate transporters, (bi)carbonate (co)transporters and carboanhydrases. Our knowledge on how pHe -signals are sensed and what the respective receptors induce inside cells is scarce. Some pH-sensitive receptors (GPR4, GPR65/TDAG8, GPR68/OGR1, GPR132/G2A, possibly GPR31 and GPR151) and ion channels (acid-sensing ion channels ASICs, transient receptor potential vanilloid receptors TRPVs) transduce signals inside cells. As little is known on the expression and function of these pH sensors, we used immunostainings to study tissue samples from common and rare skin cancers. Our current and future work is directed towards investigating the impact of all the pH-sensing receptors in different skin tumors using cell culture techniques with selective knockdown/knockout (siRNA/CRISPR-Cas9). To study cell migration and proliferation, novel impedance-based wound healing assays have been developed and are used. The field of pH sensing in tumors and wounds holds great promise for the development of pH-targeting therapies, either against pH regulators or sensors to inhibit cell proliferation and migration.

PETER-assay: Combined Impedimetric Detection of Permeability (PE) and Resistance (TER) of Barrier-Forming Cell Layers.

Epithelial and endothelial barrier function is typically studied in vitro by growing the cells of interest on permeable supports that are sandwiched between two fluid compartments. This setup mimics the physiological situation with the cell layer as the diffusion barrier at the interface between two chemically distinct fluids. Routinely, the barrier function is quantitatively described by two key parameters: (i) the transepithelial or transendothelial electrical resistance (TER) as a measure of the permeability for small inorganic ions and (ii) the permeability coefficient (PE) as a descriptor of the permeability for molecular tracers. So far the two parameters have been determined in separate experiments. This study introduces a device that allows for simultaneous detection of PE and TER of the very same cell monolayer in one single experiment (PETER-assay). The novel approach is entirely based on AC impedance measurements in two different modes, so that TER and PE become available in real time. The new approach is demonstrated for three epithelial cell lines derived from the kidney (MDCK-I, MDCK-II, NRK) with very different barrier properties under stationary conditions and when challenged by barrier-breaking fungal toxin cytochalasin D. PETER provides an excellent time-resolution and completely automated data collection.

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays.

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample's impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

Cytocompatibility of Mats Prepared from Different Electrospun Polymer Nanofibers.

Mats of cytocompatible polymer fibers are needed as scaffolds in tissue engineering or as wound healing supports. Most recently, they have emerged as matrix-material to allow for in situ chemo- and biosensing inside intact tissue fragments or surrogates. Electrospinning of such fibers from polymer solutions provides extended options to control the structural and functional properties of the resulting fiber mats. We have prepared electrospun polymeric fiber mats from poly(lactic acid) (PLA), polystyrene (PS), and poly(vinyl pyrrolidone) (PVP) with two different fiber densities. Mats and individual fibers were characterized with respect to their dimensions, morphology, and their compatibility with human keratinocytes (HaCaT) selected as a biological model. Microscopic inspection revealed that HaCaT cells were viable on mats from all three polymers with only a negligible fraction of dead cells, similar to planar control surfaces. Growth in the presence of the fiber mats did not alter cellular metabolism (ATP, redox state) and did not induce significant production of cytokines (interleukin-6 (IL-6); monocyte chemoattractant protein-1 (MCP-1)). However, we did observe that fiber density changed the overall topography of the resulting mats and led to differences in the establishment of continuous cell sheets. In conclusion, the findings support the suitability of electrospun polymeric fiber mats made from PLA, PS, or PVP as potential biocompatible matrices for future two-dimensional (2D) or three-dimensional (3D) sensing of vital parameters from tissue in health and disease.

Oxidative Stress Increases Endogenous Complement-Dependent Inflammatory and Angiogenic Responses in Retinal Pigment Epithelial Cells Independently of Exogenous Complement Sources.

Oxidative stress-induced damage of the retinal pigment epithelium (RPE) and chronic inflammation have been suggested as major contributors to a range of retinal diseases. Here, we examined the effects of oxidative stress on endogenous complement components and proinflammatory and angiogenic responses in RPE cells. ARPE-19 cells exposed for 1-48 h to H2O2 had reduced cell-cell contact and increased markers for epithelial-mesenchymal transition but showed insignificant cell death. Stressed ARPE-19 cells increased the expression of complement receptors CR3 (subunit CD11b) and C5aR1. CD11b was colocalized with cell-derived complement protein C3, which was present in its activated form in ARPE-19 cells. C3, as well as its regulators complement factor H (CFH) and properdin, accumulated in the ARPE-19 cells after oxidative stress independently of external complement sources. This cell-associated complement accumulation was accompanied by increased nlrp3 and foxp3 expression and the subsequently enhanced secretion of proinflammatory and proangiogenic factors. The complement-associated ARPE-19 reaction to oxidative stress, which was independent of exogenous complement sources, was further augmented by the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib. Our results indicate that ARPE-19 cell-derived complement proteins and receptors are involved in ARPE-19 cell homeostasis following oxidative stress and should be considered as targets for treatment development for retinal degeneration.

Tracking Hyaluronan: Molecularly Imprinted Polymer Coated Carbon Dots for Cancer Cell Targeting and Imaging.

War against cancer constantly requires new affinity tools to selectively detect, localize, and quantify biomarkers for diagnosis or prognosis. Herein, carbon nanodots (CDs), an emerging class of fluorescent nanomaterials, coupled with molecularly imprinted polymers (MIPs), are employed as a biocompatible optical imaging tool for probing cancer biomarkers. First, N-doped CDs were prepared by hydrothermal synthesis using starch as carbon source and l-tryptophan as nitrogen atom provider to achieve a high quantum yield of 25.1 ± 2%. The CDs have a typical size of ∼3.2 nm and produce an intense fluorescence at 450 nm upon excitation with UV light. A MIP shell for specific recognition of glucuronic acid (GlcA) was then synthesized around the CDs, using the emission of the CDs as an internal light source for photopolymerization. GlcA is a substructure (epitope) of hyaluronan, a biomarker for certain cancers. The biotargeting and bioimaging of hyaluronan on fixated human cervical cancer cells using CD core-MIP shell nanocomposites is demonstrated. Human keratinocytes were used as noncancerous reference cells and indeed, less staining was observed by the CD-MIP.

Label-free profiling of cell dynamics: A sequence of impedance-based assays to estimate tumor cell invasiveness in vitro.

Dynamic properties of cancer cells, most notably their ability to migrate, have been correlated successfully with their invasive nature in vivo. To establish a stronger experimental basis for such a correlation we subjected five different cancer cell lines of well-defined metastatic potential to a sequence of three independent assays reporting on three different aspects of cell dynamics, namely (1) the kinetics of cell spreading, (2) cell shape fluctuations, and (3) cell migration. The sequentially applied assays correspond to different measuring modes of the well-established ECIS technique that is based on non-invasive and label-free impedance readings of planar gold-film electrodes that serve as the growth substrate for the cells under study. Every individual assay returned a characteristic parameter describing the behavior of the cell lines in that particular assay quantitatively. The parameters of all three assays were ranked to establish individual profiles of cell dynamics for every cell line that correlate favorably with the cells' invasive properties. The sequence of impedance-based assays described here requires only small cell populations (< 10.000 cells), it is highly automated and easily adapted to 96-well formats. It provides an in-depth dynamic profile of adherent cells that might be useful in other areas besides cancer research as well.

Independent impedimetric analysis of two cell populations co-cultured on opposite sides of a porous support.

The transepithelial or -endothelial electrical resistance (TEER) is a very common and routinely recorded parameter describing the expression of barrier-forming cell-cell contacts (tight junctions) in quantitative terms. To determine TEER, barrier-forming cell monolayers are cultured on porous filter supports that separate two fluid compartments. The frequency-dependent impedance of the cell layer is then recorded and analyzed by means of equivalent circuit modelling providing TEER and the cell layer capacitance. The latter serves as a quantitative indicator for membrane topography. When cells are co-cultured on opposite sides of such a porous support to model more complex biological barriers, TEER readings will integrate over both cell layers and the individual contributions are not assessable. This study describes the modification of commonly used porous filter inserts by coating their backside with a thin gold-film. When this gold-film is used as an additional electrode, both cell layers can be studied separately by impedance analysis. The electrical parameters of either cell layer are assessable independently by switching between different electrode combinations. The performance of this new approach is illustrated and documented by experiments that (i) follow the de novo formation of cell junctions between initially suspended cells and (ii) the manipulation of mature cell-cell junctions by cytoskeleton-active drugs. Both assays confirm that both cell layers are monitored entirely independently.

Pitfalls in assessing microvascular endothelial barrier function: impedance-based devices versus the classic macromolecular tracer assay.

The most frequently used parameters to describe the barrier properties of endothelial cells (ECs) in vitro are (i) the macromolecular permeability, indicating the flux of a macromolecular tracer across the endothelium, and (ii) electrical impedance of ECs grown on gold-film electrodes reporting on the cell layer's tightness for ion flow. Due to the experimental differences between these approaches, inconsistent observations have been described. Here, we present the first direct comparison of these assays applied to one single cell type (human microvascular ECs) under the same experimental conditions. The impact of different pharmacological tools (histamine, forskolin, Y-27632, blebbistatin, TRAP) on endothelial barrier function was analyzed by Transwell(®) tracer assays and two commercial impedance devices (xCELLigence(®), ECIS(®)). The two impedance techniques provided very similar results for all compounds, whereas macromolecular permeability readings were found to be partly inconsistent with impedance. Possible reasons for these discrepancies are discussed. We conclude that the complementary combination of both approaches is highly recommended to overcome the restrictions of each assay. Since the nature of the growth support may contribute to the observed differences, structure-function relationships should be based on cells that are consistently grown on either permeable or impermeable growth supports in all experiments.

Cell-Based Microarrays for In Vitro Toxicology.

DNA/RNA and protein microarrays have proven their outstanding bioanalytical performance throughout the past decades, given the unprecedented level of parallelization by which molecular recognition assays can be performed and analyzed. Cell microarrays (CMAs) make use of similar construction principles. They are applied to profile a given cell population with respect to the expression of specific molecular markers and also to measure functional cell responses to drugs and chemicals. This review focuses on the use of cell-based microarrays for assessing the cytotoxicity of drugs, toxins, or chemicals in general. It also summarizes CMA construction principles with respect to the cell types that are used for such microarrays, the readout parameters to assess toxicity, and the various formats that have been established and applied. The review ends with a critical comparison of CMAs and well-established microtiter plate (MTP) approaches.