Highly scalable and standardized organ-on-chip platform with TEER for biological barrier modeling.

The development of new therapies is hampered by the lack of predictive, and patient-relevant in vitro models. Organ-on-chip (OOC) technologies can potentially recreate physiological features and hold great promise for tissue and disease modeling. However, the non-standardized design of these chips and perfusion control systems has been a barrier to quantitative high-throughput screening (HTS). Here we present a scalable OOC microfluidic platform for applied kinetic in vitro assays (AKITA) that is applicable for high, medium, and low throughput. Its standard 96-well plate and 384-well plate layouts ensure compatibility with existing laboratory workflows and high-throughput data collection and analysis tools. The AKITA plate is optimized for the modeling of vascularized biological barriers, primarily the blood-brain barrier, skin, and lung, with precise flow control on a custom rocker. The integration of trans-epithelial electrical resistance (TEER) sensors allows rapid and repeated monitoring of barrier integrity over long time periods. Together with automated liquid handling and compound permeability testing analyses, we demonstrate the flexibility of the AKITA platform for establishing human-relevant models for preclinical drug and precision medicine's efficacy, toxicity, and permeability under near-physiological conditions.

Cellular Models of Alpha-Synuclein Aggregation: What Have We Learned and Implications for Future Study.

Alpha-synuclein's role in diseases termed "synucleinopathies", including Parkinson's disease, has been well-documented. However, after over 25 years of research, we still do not fully understand the alpha-synuclein protein and its role in disease. In vitro cellular models are some of the most powerful tools that researchers have at their disposal to understand protein function. Advantages include good control over experimental conditions, the possibility for high throughput, and fewer ethical issues when compared to animal models or the attainment of human samples. On the flip side, their major disadvantages are their questionable relevance and lack of a "whole-brain" environment when it comes to modeling human diseases, such as is the case of neurodegenerative disorders. Although now, with the advent of pluripotent stem cells and the ability to create minibrains in a dish, this is changing. With this review, we aim to wade through the recent alpha-synuclein literature to discuss how different cell culture setups (immortalized cell lines, primary neurons, human induced pluripotent stem cells (hiPSCs), blood-brain barrier models, and brain organoids) can help us understand aggregation pathology in Parkinson's and other synucleinopathies.

Toward Full-Stack In Silico Synthetic Biology: Integrating Model Specification, Simulation, Verification, and Biological Compilation.

We present the Infobiotics Workbench (IBW), a user-friendly, scalable, and integrated computational environment for the computer-aided design of synthetic biological systems. It supports an iterative workflow that begins with specification of the desired synthetic system, followed by simulation and verification of the system in high-performance environments and ending with the eventual compilation of the system specification into suitable genetic constructs. IBW integrates modeling, simulation, verification, and biocompilation features into a single software suite. This integration is achieved through a new domain-specific biological programming language, the Infobiotics Language (IBL), which tightly combines these different aspects of in silico synthetic biology into a full-stack integrated development environment. Unlike existing synthetic biology modeling or specification languages, IBL uniquely blends modeling, verification, and biocompilation statements into a single file. This allows biologists to incorporate design constraints within the specification file rather than using decoupled and independent formalisms for different in silico analyses. This novel approach offers seamless interoperability across different tools as well as compatibility with SBOL and SBML frameworks and removes the burden of doing manual translations for standalone applications. We demonstrate the features, usability, and effectiveness of IBW and IBL using well-established synthetic biological circuits.

Utilising Induced Pluripotent Stem Cells in Neurodegenerative Disease Research: Focus on Glia.

Induced pluripotent stem cells (iPSCs) are a self-renewable pool of cells derived from an organism's somatic cells. These can then be programmed to other cell types, including neurons. Use of iPSCs in research has been two-fold as they have been used for human disease modelling as well as for the possibility to generate new therapies. Particularly in complex human diseases, such as neurodegenerative diseases, iPSCs can give advantages over traditional animal models in that they more accurately represent the human genome. Additionally, patient-derived cells can be modified using gene editing technology and further transplanted to the brain. Glial cells have recently become important avenues of research in the field of neurodegenerative diseases, for example, in Alzheimer's disease and Parkinson's disease. This review focuses on using glial cells (astrocytes, microglia, and oligodendrocytes) derived from human iPSCs in order to give a better understanding of how these cells contribute to neurodegenerative disease pathology. Using glia iPSCs in in vitro cell culture, cerebral organoids, and intracranial transplantation may give us future insight into both more accurate models and disease-modifying therapies.

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils.

BACKGROUND: Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. RESULTS: Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. CONCLUSIONS: Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.

On the importance of modelling the internal spatial dynamics of biological cells.

Spatial effects such as cell shape have very often been considered negligible in models of cellular pathways, and many existing simulation infrastructures do not take such effects into consideration. Recent experimental results are reversing this judgement by showing that very small spatial variations can make a big difference in the fate of a cell. This is particularly the case when considering eukaryotic cells, which have a complex physical structure and many subtle control mechanisms, but bacteria are also interesting for the huge variation in shape both between species and in different phases of their lifecycle. In this work we perform simulations that measure the effect of three common bacterial shapes on the behaviour of model cellular pathways. To perform these experiments we develop ReDi-Cell, a highly scalable GPGPU cell simulation infrastructure for the modelling of cellular pathways in spatially detailed environments. ReDi-Cell is validated against known-good simulations, prior to its use in new work. We then use ReDi-Cell to conduct novel experiments that demonstrate the effect that three common bacterial shapes (Cocci, Bacilli and Spirilli) have on the behaviour of model cellular pathways. Pathway wavefront shape, pathway concentration gradients, and chemical species distribution are measured in the three different shapes. We also quantify the impact of internal cellular clutter on the same pathways. Through this work we show that variations in the shape or configuration of these common cell shapes alter model cell behaviour.

Spatial simulations of myxobacterial development.

Many bacteria exhibit multicellular behaviour, with individuals within a colony coordinating their actions for communal benefit. One example of complex multicellular phenotypes is myxobacterial fruiting body formation, where thousands of cells aggregate into large three-dimensional structures, within which sporulation occurs. Here we describe a novel theoretical model, which uses Monte Carlo dynamics to simulate and explain multicellular development. The model captures multiple behaviours observed during fruiting, including the spontaneous formation of aggregation centres and the formation and dissolution of fruiting bodies. We show that a small number of physical properties in the model is sufficient to explain the most frequently documented population-level behaviours observed during development in Myxococcus xanthus.