Assessing the performance of the Cell Painting assay across different imaging systems.

Quantitative microscopy is a powerful method for performing phenotypic screens from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments. Here, we examine the performance of the Cell Painting assay across multiple high-throughput microscope systems and find that all are compatible with this assay. Furthermore, we determine independently for each microscope system the best performing settings, providing those who wish to adopt this assay an ideal starting point for their own assays. We also explore the impact of microscopy setting changes in the Cell Painting assay and find that few dramatically reduce the quality of a Cell Painting profile, regardless of the microscope used.

Assessing the performance of the Cell Painting assay across different imaging systems.

Quantitative microscopy is a powerful method for performing phenotypic screens from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments. Here, we examine the performance of the Cell Painting assay across multiple high-throughput microscope systems and find that all are compatible with this assay. Furthermore, we determine independently for each microscope system the best performing settings, providing those who wish to adopt this assay an ideal starting point for their own assays. We also explore the impact of microscopy setting changes in the Cell Painting assay and find that few dramatically reduce the quality of a Cell Painting profile, regardless of the microscope used.

A Framework for Optimizing High-Content Imaging of 3D Models for Drug Discovery.

Three-dimensional (3D) spheroid models are rapidly gaining favor for drug discovery applications due to their improved morphological characteristics, cellular complexity, long lifespan in culture, and higher physiological relevance relative to two-dimensional (2D) cell culture models. High-content imaging (HCI) of 3D spheroid models has the potential to provide valuable information to help researchers untangle disease pathophysiology and assess novel therapies more effectively. The transition from 2D monolayer models to dense 3D spheroids in HCI applications is not trivial, however, and requires 3D-optimized protocols, instrumentation, and resources. Here, we discuss considerations for moving from 2D to 3D models and present a framework for HCI and analysis of 3D spheroid models in a drug discovery setting. We combined scaffold-free, multicellular spheroid models with scalable, automation-compatible plate technology enabling image-based applications ranging from high-throughput screening to more complex, lower-throughput microphysiological systems of organ networks. We used this framework in three case studies: investigation of lipid droplet accumulation in a human liver nonalcoholic steatohepatitis (NASH) model, real-time immune cell interactions in a multicellular 3D lung cancer model, and a high-throughput screening application using a 3D co-culture model of gastric carcinoma to assess dose-dependent drug efficacy and specificity. The results of these proof-of-concept studies demonstrate the potential for high-resolution image-based analysis of 3D spheroid models for drug discovery applications, and confirm that cell-level and temporal-spatial analyses that fully exploit multicellular features of spheroid models are not only possible but soon will be routine practice in drug discovery workflows.

Influence of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on ubiquinone levels in rat skeletal muscle and heart: relationship to cytotoxicity and inhibitory activity for cholesterol synthesis in human skeletal muscle cells.

Although statins are prescribed as relatively safe and effective drugs for hypercholesterolemic patients, it has been reported that a significant side effect, myopathy, occurs infrequently during medication. Moreover, because statins decrease cardiac ubiquinone levels, the risk of cardiac dysfunction has been suggested. This study sought to evaluate and compare the cytotoxicity of statins (cerivastatin, pitavastatin, fluvastatin, simvastatin, atorvastatin and pravastatin) in cultured human skeletal muscle cells (HSkMCs) and the effects on ubiquinone levels in statin-treated rat skeletal muscle and heart. Cerivastatin, the most potent inhibitor of HMG-CoA reductase, showed the strongest cytotoxicity (over 10-fold) among the statins examined, while the effects of the others were in a similar range. In rat experiments, neither pitavastatin nor cerivastatin decreased ubiquinone levels in skeletal muscle, but both dose-dependently lowered ubiquinone levels in the heart. As the rates of reduction by pitavastatin (9.6% at 30 mg/kg) and cerivastatin (9.7% at 0.3 mg/kg) were almost equal, it was estimated that cerivastatin reduced ubiquinone levels in the rat heart approximately 100-fold more strongly than pitavastatin, based on the effective doses. We found that cerivastatin showed the most potent cytotoxicity in HSkMCs and strongly lowered ubiquinone levels in the rat heart.

[Use of magnesium sulfate during resection of pheochromocytoma].

A 75-year-old woman was scheduled to undergo resection of pheochromocytoma under general and epidural anesthesia. Continuous infusion of magnesium sulfate was initiated at the time of tracheal intubation and was terminated at the tumor resection. Intraoperative blood pressure and heart rate were stable, but blood pressure rose above 160 mmHg when the tumor was handled. Hypertension caused by the tumor manipulation was successfully treated with intravenous nicardipine. Following the tumor removal, reduced blood pressure was treated with dopamine and norepinephrine. After the operation, spontaneous respiration did not appear until 120 minutes following the last vecuronium injection. Although neuromuscular blockade was reversed with neostigmine and atropine, muscle tone was not restored and satisfactory spontaneous respiration was not obtained. One hour later the patient was extubated. Intraoperative use of magnesium sulfate provides adequate hemodynamic stability for resection of pheochromocytoma, but may cause prolonged neuromuscular blockade. Monitoring of neuromuscular function should be essential and reduction of ve curonium dose should be considered on using magnesium sulfate intraoperatively.