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 product 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

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

Abstract 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

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