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Background and Aims: To better understand nonalcoholic steatohepatitis (NASH) disease progression and to evaluate drug targets and compound activity, we undertook the development of an in vitro 3D model to mimic liver architecture and the NASH environment. Methods: We have developed an in vitro preclinical 3D NASH model by coculturing primary human hepatocytes, human stellate cells, liver endothelial cells and Kupffer cells embedded in a hydrogel of rat collagen on a 96-well plate. A NASH-like environment was induced by addition of medium containing free fatty acids and tumor necrosis factor-α. This model was then characterized by biochemical, imaging and transcriptomics analyses. Results: We succeeded in defining suitable culture conditions to maintain the 3D coculture for up to 10 days in vitro, with the lowest level of steatosis and reproducible low level of inflammation and fibrosis. NASH disease was induced with a custom medium mimicking NASH features. The cell model exhibited the key NASH disease phenotypes of hepatocyte injury, steatosis, inflammation, and fibrosis. Hepatocyte injury was highlighted by a decrease of CYP3A4 expression and activity, without loss of viability up to day 10. Moreover, the model was able to stimulate a stable inflammatory and early fibrotic environment, with expression and secretion of several cytokines. A global gene expression analysis confirmed the NASH induction. Conclusions: This is a new in vitro model of NASH disease consisting of four human primary cell-types that exhibits most features of the disease. The 10-day cell viability and cost effectiveness of the model make it suitable for medium throughput drug screening and provide attractive avenues to better understand disease physiology and to identify and characterize new drug targets.

The LabEx Milieu Interieur (MI) project is a clinical study centered on the detailed characterization of the baseline and induced immune responses in blood samples from 1,000 healthy donors. Analyses of these samples has lay ground for seminal studies on the genetic and environmental determinants of immunologic variance in a healthy cohort population. In the current study we developed in vitro methods enabling standardized quantification of MI-cohort-derived primary fibroblasts responses. Our results show that in vitro human donor cohort fibroblast responses to stimulation by different MAMPs analogs allows to characterize individual donor immune-phenotype variability. The results provide proof-of-concept foundation to a new experimental framework for such studies. A bio-bank of primary fibroblast lines was generated from 323 out of 1,000 healthy individuals selected from the MI-study cohort. To study inter-donor variability of innate immune response in primary human dermal fibroblasts we chose to measure the TLR3 and TLR4 response pathways, both receptors being expressed and previously studied in fibroblasts. We established high-throughput automation compatible methods for standardized primary fibroblast cell activation, using purified MAMPS analogs, poly I:C and LPS that stimulate TLR3 and TLR4 pathways respectively. These results were in turn compared with a stimulation method using infection by HSV-1 virus. Our “Add-only” protocol minimizes high-throughput automation system variability facilitating whole process automation from cell plating through stimulation to recovery of cell supernatants, and fluorescent labeling. Images were acquired automatically by high-throughput acquisition on an automated high-content imaging microscope. Under these methodological conditions standardized image acquisition provided for quantification of cellular responses allowing biological variability to be measured with low system noise and high biological signal fidelity. Optimal for automated analysis of immuno-phenotype of primary human cell responses our method and experimental framework as reported here is highly compatible to high-throughput screening protocols like those necessary for chemo-genomic screening. In context of primary fibroblasts derived from donors enrolled to the MI-clinical-study our results open the way to assert the utility of studying immune-phenotype characteristics relevant to a human clinical cohort.

CdSe quantum dots (QDs) are valuable tools for deciphering molecular mechanisms in cells. Their conjugation with antibodies offers a unique staining source with optimal characteristics, including increased photostability and narrow emission spectra, allowing for improved multiplexing capabilities using a single excitation source. In combination with pathology models derived from patients, they have great potential to contribute to quantitative molecular profiling and promote personalized medicine. However, the commercial availability of diverse CdSe QDs is still limited and characterization techniques must be performed to these materials or the conjugates developed in the lab to assure a proper function and reproducibility. Furthermore, while there is significant data of QDs experiments in cell lines, the literature with primary human cells is scarce, and QD behavior in these systems may be different. Rigorous characterization data of commercially available QDs and their conjugates with biomolecules of interest is needed in order to establish their potential for target labelling and expand their use among research labs. Here we compare the characterization and labelling performance of different QD conjugates in SH-SY5Y cell line, fibroblasts and immortalized lymphocytes derived from amyotrophic lateral sclerosis patients.

Lung exposure to inhaled particulate matter (PM) is known to injure the airway epithelium via inflammation, a phenomenon linked to increased levels of global morbidity and mortality. To evaluate physiological outcomes following PM exposure and concurrently circumvent the use of animal experiments, in vitro approaches have typically relied on traditional assays with plates or well inserts. Yet, these manifest drawbacks including the inability to capture physiological inhalation conditions and aerosol deposition characteristics relative to in vivo human conditions. Here, we present a novel airway-on-chip exposure platform that emulates the epithelium of human bronchial airways with critical cellular barrier functions at an air-liquid interface (ALI). As a proof-of-concept for in vitro lung cytotoxicity testing, we recapitulate a well-characterized cell apoptosis pathway, induced through exposure to 2 μm airborne particles coated with αVR1 antibody that leads to significant loss in cell viability across the recapitulated airway epithelium. Notably, our in vitro inhalation assays enable simultaneous aerosol exposure across multiple airway chips integrated within a larger bronchial airway tree model, under physiological respiratory airflow conditions. Our findings underscore in situ-like aerosol deposition outcomes where patterns depend on respiratory flows across the airway tree geometry and gravitational orientation, as corroborated by concurrent numerical simulations. Our airway-on-chips not only highlight the prospect of realistic in vitro exposure assays in recapitulating characteristic local in vivo deposition outcomes, such platforms open opportunities toward advanced in vitro exposure assays for preclinical cytotoxicity and drug screening applications.

International audience; In this article, we first developed a new medium to culture together primary human osteoblastic, osteoclastic, and endothelial cells (ECs) chosen to represent the three major bone cell tissues. Indeed, no study has been conducted on primary human cells and on the phenotype/activity retention of these three primary human cell types. Thus, we established an original triculture model with osteoblastic, osteoclastic, and ECs, where not only both cell phenotype and cell activity were maintained but also cell culture homeostasis. These promising results will permit further investigations to create in vitro conditions to mimic the bone microenvironment and analyze cell interactions in ex vivo studies.

In this paper, we present a comprehensive discussion of the results obtained after in vitro exposure of human fetal fibroblasts and human adult fibroblasts to pulsed radiation in a wide band between 100 and 150 GHz and to continuous wave radiation at 25 GHz. In order to assess potential effects of exposure, the genome integrity, cell cycle, cytological ultrastructure, and proteins expression were evaluated.

Cabozantinib, a c-MET and vascular endothelial growth factor receptor 2 inhibitor, demonstrated to prolong progression free survival and improve skeletal disease-related endpoints in castration-resistant prostate cancer and in metastatic renal carcinoma. Our purpose is to investigate the direct effect of cabozantinib on bone microenvironment using a total human model of primary osteoclasts and osteoblasts.Osteoclasts were differentiated from monocytes isolated from healthy donors; osteoblasts were derived from human mesenchymal stem cells obtained from bone fragments of orthopedic surgery patients. Osteoclast activity was evaluated by tartrate resistant acid phosphatase (TRAP) staining and bone resorption assays and osteoblast differentiation was detected by alkaline phosphatase and alizarin red staining.Our results show that non-cytotoxic doses of cabozantinib significantly inhibit osteoclast differentiation (p=0.0145) and bone resorption activity (p=0.0252). Moreover, cabozantinib down-modulates the expression of osteoclast marker genes, TRAP (p=0.006), CATHEPSIN K (p=0.004) and Receptor Activator of Nuclear Factor k B (RANK) (p=0.001). Cabozantinib treatment has no effect on osteoblast viability or differentiation, but increases osteoprotegerin mRNA (p=0.015) and protein levels (p=0.004) and down-modulates Receptor Activator of Nuclear Factor k B Ligand (RANKL) at both mRNA (p, info:eu-repo/semantics/published

In this work we present biological effects of silver nanoparticles (AgNPs) synthesized by picosecond laser ablation of silver in deionized water. We examined induction of chromosomal aberrations, lymphocyte micronuclei, appearance and recovery of double strand breaks (DSBs) of DNA, cell proliferation potential, concentration of lipid peroxidation products and insulin-like growth factor 1 (ILGF-1). We found that AgNPs sized from 3 nm to 8 nm induce cell cytostasis, which is accompanied with its clastogenic action on DNA, while AgNPs, sized 2 nm behaves contrary stimulating cell proliferation by enhancing ILGF-1 concentration. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Introduction Human primary cells originating from different locations within the body could differ greatly in their metabolic phenotypes, influencing both how they act during physiological/pathological processes and how susceptible/resistant they are to a variety of disease risk factors. A novel way to monitor cellular metabolism is through cell energetics assays, so we explored this approach with human primary cell types, as models of sclerotic disorders. Objectives In order to better understand pathophysiological processes at the cellular level, our goals were to measure metabolic pathway activities of endothelial cells and fibroblasts, and determine their metabolic phenotype profiles. Methods Biolog Phenotype MicroArray™ technology was used for the first time to characterize metabolic phenotypes of diverse primary cells. These colorimetric assays enable detection of utilization of 367 specific biochemical substrates by human endothelial cells from the coronary artery (HCAEC), umbilical vein (HUVEC) and normal, healthy lung fibroblasts (NHLF). Results Adenosine, inosine, d-mannose and dextrin were strongly utilized by all three cell types, comparable to glucose. Substrates metabolized solely by HCAEC were mannan, pectin, gelatin and prevalently tricarballylic acid. HUVEC did not show any uniquely metabolized substrates whereas NHLF exhibited strong utilization of sugars and carboxylic acids along with amino acids and peptides. Conclusion Taken together, we show for the first time that this simple energetics assay platform enables metabolic characterization of primary cells and that each of the three human cell types examined gives a unique and distinguishable profile.