Glutamatergic synaptic input to glioma cells drives brain tumour progression.

A network of communicating tumour cells that is connected by tumour microtubes mediates the progression of incurable gliomas. Moreover, neuronal activity can foster malignant behaviour of glioma cells by non-synaptic paracrine and autocrine mechanisms. Here we report a direct communication channel between neurons and glioma cells in different disease models and human tumours: functional bona fide chemical synapses between presynaptic neurons and postsynaptic glioma cells. These neurogliomal synapses show a typical synaptic ultrastructure, are located on tumour microtubes, and produce postsynaptic currents that are mediated by glutamate receptors of the AMPA subtype. Neuronal activity including epileptic conditions generates synchronised calcium transients in tumour-microtube-connected glioma networks. Glioma-cell-specific genetic perturbation of AMPA receptors reduces calcium-related invasiveness of tumour-microtube-positive tumour cells and glioma growth. Invasion and growth are also reduced by anaesthesia and the AMPA receptor antagonist perampanel, respectively. These findings reveal a biologically relevant direct synaptic communication between neurons and glioma cells with potential clinical implications.

Comparison of cell viability methods for human mesenchymal/stromal stem cells and human A549 lung carcinoma cells after freeze-thaw stress.

For the safety and efficacy of frozen cell therapy products, determination of cellular viability is key. However, results of cell viability measurements do not only depend on the cell line or on the inflicted stress, but also on the assay used, making inter-experimental comparisons difficult. The aim of this study was thus to assess commonly used viability assays in clinically relevant human mesenchymal/stromal stem cells and human A549 lung carcinoma cells. Post freeze-thaw stress viability and proliferation were evaluated under different conditions using trypan blue, acridine orange/DAPI stain, alamarBlue, ATP, and neutral red assays. Significant differences in cell viability between metabolic assays were observed, likely due to their distinct intrinsic detection mechanisms. Membrane-integrity based assays generally overestimated cell viabilities in this study. Furthermore, noticeable differences in inter-assay sensitivities were observed. These differences highlight that cell viability methods should be meticulously selected and their associated results carefully interpreted in a relevant context to ensure reliable conclusions. Indeed, although cell membrane integrity based assays are a popular choice to determine cellular quality attributes after freezing and thawing, we demonstrate that metabolic assays may be more suitable in this context.

Cryoprotection in Human Mesenchymal Stromal/Stem Cells: Synergistic Impact of Urea and Glucose.

Standard freezing protocols of clinically relevant cell lines commonly employ agents such as fetal bovine serum and dimethyl sulfoxide, which are a potential concern from both a regulatory and a patient safety perspective. The aim of this work was to develop formulations with safe and well tolerated excipients for the (cryo-) preservation of cell therapy products. We evaluated the cryoprotective capabilities of urea and glucose through measurements of cell metabolic activity. Freezing of clinically relevant human mesenchymal stromal/stem cells and human dermal fibroblasts at ≤ - 65°C at equimolar ratios of urea and glucose resulted in comparable viabilities to established dimethyl sulfoxide. Pre-incubation of human mesenchymal stromal/stem cells in trehalose and addition of mannitol and sucrose to the formulation further enhanced cell viability after freeze-thaw stress. Other cell types assessed (A549 and SK-N-AS) could not satisfactorily be preserved with urea and glucose, highlighting the need for tailored formulations to sustain acceptable cryopreservation.

Challenges for Cell-Based Medicinal Products From a Pharmaceutical Product Perspective.

Advanced therapy medicinal products (ATMPs), such as somatic cell-therapy medicinal products or tissue-engineered products for human use, offer new and potentially curative opportunities to treat yet untreatable diseases or disorders. For cell-therapy medicinal products (CBMPs), multiple stability and quality challenges exist and relate to the cellular composition and unstable nature of these parenteral preparations. It is the aim of this review to discuss open questions and problems associated with the development, manufacturing and testing of CBMPs from a pharmaceutical drug product perspective. This includes safety, storage and handling, particulates, the choice of container closure systems and integrity. Analytical methods commonly used to evaluate the quality of the final CBMP to ensure patient's safety will be discussed. Particulate contamination in final products deserve special attention since CBMPs cannot be sterile filtered. Visible and sub-visible particles may represent environmental contaminations or may form during storage. They may be introduced from processing materials such as single use product contact materials, ancillary materials, or any components such as primary packaging used for the final product. Currently available analytical methods for detecting particulates may not be easily applicable to CBMPs due to their inherent particulate nature and appearance.

Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins.

With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the 'labeling barrier' and to bypass photobleaching in multi-plane, whole-cell 3D experiments.