A spheroid whole mount drug testing pipeline with machine-learning based image analysis identifies cell-type specific differences in drug efficacy on a single-cell level.

Background: The growth and drug response of tumors are influenced by their stromal composition, both in vivo and 3D-cell culture models. Cell-type inherent features as well as mutual relationships between the different cell types in a tumor might affect drug susceptibility of the tumor as a whole and/or of its cell populations. However, a lack of single-cell procedures with sufficient detail has hampered the automated observation of cell-type-specific effects in three-dimensional stroma-tumor cell co-cultures.

A multiparametric analysis including single-cell and subcellular feature assessment reveals differential behavior of spheroid cultures on distinct ultra-low attachment plate types.

Spheroids have become principal three-dimensional models to study cancer, developmental processes, and drug efficacy. Single-cell analysis techniques have emerged as ideal tools to gauge the complexity of cellular responses in these models. However, the single-cell quantitative assessment based on 3D-microscopic data of the subcellular distribution of fluorescence markers, such as the nuclear/cytoplasm ratio of transcription factors, has largely remained elusive.

Aberrant evoked calcium signaling and nAChR cluster morphology in a D90A hiPSC-derived neuromuscular model.

Familial amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder that is due to mutations in one of several target genes, including . So far, clinical records, rodent studies, and models have yielded arguments for either a primary motor neuron disease, or a pleiotropic pathogenesis of ALS. While mouse models lack the human origin, models using human induced pluripotent stem cells (hiPSC) have been recently developed for addressing ALS pathogenesis.

Quantitative Analysis of Whole-Mount Fluorescence-Stained Tumor Spheroids in Phenotypic Drug Screens.

Three-dimensional cell cultures, such as spheroids or organoids, serve as important models for drug screening purposes. Optical tissue clearing (OTC) enhances the visualization of fluorescence stainings and enables in toto microscopy of 3D cell culture models. Furthermore, subsequent automated image analysis tools convert qualitative confocal image sets into quantitative data.

Schwann cells in neuromuscular models.

Neuromuscular cell culture models are used to investigate synapse formation and function, as well as mechanisms of de-and regeneration in neuromuscular diseases. Recent developments including 3D culture technique and hiPSC technology have propelled their ability to complement insights from models. However, most cultures have not considered Schwann cells, the glial part of NMJs.

Two peak elution behavior of a monoclonal antibody in cation exchange chromatography as a screening tool for excipients.

Aggregation of proteins is a critical quality attribute and a major concern during the purification of therapeutic proteins, like monoclonal antibodies. In-solution experiments applying different stress scenarios, e.g.

Development and In Vitro Differentiation of Schwann Cells.

Schwann cells are glial cells of the peripheral nervous system. They exist in several subtypes and perform a variety of functions in nerves. Their derivation and culture in vitro are interesting for applications ranging from disease modeling to tissue engineering.

Application of the Steric Mass Action formalism for modeling under high loading conditions: Part 1. Investigation of the influence of pH on the steric shielding factor.

In ion exchange chromatography, the Steric Mass Action (SMA) formalism is frequently used to simulate sorption processes at low and high column load conditions. To apply the SMA model for describing protein elution over wide ranges of pH, it is necessary to use pH-dependent model parameters. In the past, some publications have already described the pH-dependence of the characteristic protein charge and the equilibrium constant, while the influence of pH on the steric shielding factor has been mostly neglected.

Mechanistic modeling and simulation of a complex low and high loading elution behavior of a polypeptide in cation exchange chromatography.

The mechanistic modeling of preparative liquid chromatography is still a challenging task. Nonideal thermodynamic conditions may require activity coefficients for the mechanistic description of preparative chromatography. In this work, a chromatographic cation exchange step with a polypeptide having a complex elution behavior in low and high loading situations is modeled.

3D Melanoma Cocultures as Improved Models for Nanoparticle-Mediated Delivery of RNA to Tumors.

Cancer therapy is an emergent application for mRNA therapeutics. While in tumor immunotherapy, mRNA encoding for tumor-associated antigens is delivered to antigen-presenting cells in spleen and lymph nodes, other therapeutic options benefit from immediate delivery of mRNA nanomedicines directly to the tumor. However, tumor targeting of mRNA therapeutics is still a challenge, since, in addition to delivery of the cargo to the tumor, specifics of the targeted cell type as well as its interplay with the tumor microenvironment are crucial for successful intervention.

hiPSC-Derived Schwann Cells Influence Myogenic Differentiation in Neuromuscular Cocultures.

Motoneurons, skeletal muscle fibers, and Schwann cells form synapses, termed neuromuscular junctions (NMJs). These control voluntary body movement and are affected in numerous neuromuscular diseases. Therefore, a variety of NMJ in vitro models have been explored to enable mechanistic and pharmacological studies.

Sweet Taste Is Complex: Signaling Cascades and Circuits Involved in Sweet Sensation.

Sweetness is the preferred taste of humans and many animals, likely because sugars are a primary source of energy. In many mammals, sweet compounds are sensed in the tongue by the gustatory organ, the taste buds. Here, a group of taste bud cells expresses a canonical sweet taste receptor, whose activation induces Ca rise, cell depolarization and ATP release to communicate with afferent gustatory nerves.

Extracellular Matrix Components Regulate Bone Sialoprotein Expression in MDA-MB-231 Breast Cancer Cells.

Bone sialoprotein (BSP) has become a target in breast cancer research as it is associated with tumor progression and metastasis. The mechanisms underlying the regulation of BSP expression have been largely elusive. Given that BSP is involved in the homing of cancer cells in bone metastatic niches, we addressed regulatory effects of proteolytic cleavage and extracellular matrix components on BSP expression and distribution in cell culture models.

Regulatory Function of Sympathetic Innervation on the Endo/Lysosomal Trafficking of Acetylcholine Receptor.

Recent studies have demonstrated that neuromuscular junctions are co-innervated by sympathetic neurons. This co-innervation has been shown to be crucial for neuromuscular junction morphology and functional maintenance. To improve our understanding of how sympathetic innervation affects nerve-muscle synapse homeostasis, we here used imaging, proteomic, biochemical, and microscopic approaches to compare normal and sympathectomized mouse hindlimb muscles.

Preparation, Drug Treatment, and Immunohistological Analysis of Tri-Culture Spheroid 3D Melanoma-Like Models.

Most currently available three-dimensional melanoma models have either focused on simplicity or were optimized for physiological relevance. Accordingly, these paradigms have been either composed of malignant cells only or they were sophisticated human skin equivalents featuring multiple cell types and skin-like organization. Here, an intermediate spheroid-based assay system is presented, which uses tri-cultures of human CCD-1137Sk fibroblasts, HaCaT keratinocytes, and SK-MEL-28 melanoma cells.

Glyco-engineered HEK 293-F cell lines for the production of therapeutic glycoproteins with human N-glycosylation and improved pharmacokinetics.

N-glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N-acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N-glycans often trigger rapid clearance from the patient's bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production.

Mechanistic modeling of ligand density variations on anion exchange chromatography.

Ion exchange chromatography is a powerful and ubiquitous unit operation in the purification of therapeutic proteins. However, the performance of an ion-exchange process depends on a complex interrelationship between several parameters, such as protein properties, mobile phase conditions, and chromatographic resin characteristics. Consequently, batch variations of ion exchange resins play a significant role in the robustness of these downstream processing steps.

Compression Bioreactor-Based Mechanical Loading Induces Mobilization of Human Bone Marrow-Derived Mesenchymal Stromal Cells into Collagen Scaffolds In Vitro.

Articular cartilage (AC) is an avascular tissue composed of scattered chondrocytes embedded in a dense extracellular matrix, in which nourishment takes place via the synovial fluid at the surface. AC has a limited intrinsic healing capacity, and thus mainly surgical techniques have been used to relieve pain and improve function. Approaches to promote regeneration remain challenging.

POFUT1 mRNA expression as an independent prognostic parameter in muscle-invasive bladder cancer.

Muscle-invasive bladder cancer (MIBC) is characterized by high recurrence and rapid progression. Progression is linked to changes in glycan structures and altered levels of glycosyltransferases. The relationship of mRNA expression by glycosyltransferase genes B4GALT1, EXT1, MGAT5B, and POFUT1 to the probability of surviving MIBC after radical cystectomy has not yet been investigated.

An alternative pathway for sweet sensation: possible mechanisms and physiological relevance.

Sweet substances are detected by taste-bud cells upon binding to the sweet-taste receptor, a T1R2/T1R3 heterodimeric G protein-coupled receptor. In addition, experiments with mouse models lacking the sweet-taste receptor or its downstream signaling components led to the proposal of a parallel "alternative pathway" that may serve as metabolic sensor and energy regulator. Indeed, these mice showed residual nerve responses and behavioral attraction to sugars and oligosaccharides but not to artificial sweeteners.

A Scaffold-Free 3-D Co-Culture Mimics the Major Features of the Reverse Warburg Effect In Vitro.

Most tumors consume large amounts of glucose. Concepts to explain the mechanisms that mediate the achievement of this metabolic need have proposed a switch of the tumor mass to aerobic glycolysis. Depending on whether primarily tumor or stroma cells undergo such a commutation, the terms 'Warburg effect' or 'reverse Warburg effect' were coined to describe the underlying biological phenomena.

Effects of ASC Application on Endplate Regeneration Upon Glycerol-Induced Muscle Damage.

Amongst other approaches, adipose-derived stromal cells (ASCs) have recently been tested with respect to their regenerative capacity for treatment of neuromuscular disorders. While beneficial effects of ASCs on muscle recovery were observed previously, their impact on regeneration of neuromuscular junctions (NMJs) is unclear. Here, we used a murine glycerol damage model to study disruption and regeneration of NMJs and to evaluate the effects of systemic application of ASCs on muscle and NMJ recovery.

Sensing Senses: Optical Biosensors to Study Gustation.

The five basic taste modalities, sweet, bitter, umami, salty and sour induce changes of Ca levels, pH and/or membrane potential in taste cells of the tongue and/or in neurons that convey and decode gustatory signals to the brain. Optical biosensors, which can be either synthetic dyes or genetically encoded proteins whose fluorescence spectra depend on levels of Ca, pH or membrane potential, have been used in primary cells/tissues or in recombinant systems to study taste-related intra- and intercellular signaling mechanisms or to discover new ligands. Taste-evoked responses were measured by microscopy achieving high spatial and temporal resolution, while plate readers were employed for higher throughput screening.

Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward.

Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid- and chip-based three-dimensional cell cultures of approximately 300 μm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein.

Analysis of calcium signaling in live human Tongue cell 3D-Cultures upon tastant perfusion.

Bridging the gap between two-dimensional cell cultures and complex in vivo tissues, three-dimensional cell culture models are of increasing interest in the fields of cell biology and pharmacology. However, present challenges hamper live cell imaging of three-dimensional cell cultures. These include (i) the stabilization of these structures under perfusion conditions, (ii) the recording of many z-planes at high spatio-temporal resolution, (iii) and the data analysis that ranges in complexity from whole specimens to single cells.