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- Koutsilieri, Stefania, et al.
(författare)
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Proteomic workflows for deep phenotypic profiling of 3D organotypic liver models
- 2024
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Ingår i: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 19:3
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Tidskriftsartikel (refereegranskat)abstract
- Organotypic human tissue models constitute promising systems to facilitate drug discovery and development. They allow to maintain native cellular phenotypes and functions, which enables long-term pharmacokinetic and toxicity studies, as well as phenotypic screening. To trace relevant phenotypic changes back to specific targets or signaling pathways, comprehensive proteomic profiling is the gold-standard. A multitude of proteomic workflows have been applied on 3D tissue models to quantify their molecular phenotypes; however, their impact on analytical results and biological conclusions in this context has not been evaluated. The performance of twelve mass spectrometry-based global proteomic workflows that differed in the amount of cellular input, lysis protocols and quantification methods was compared for the analysis of primary human liver spheroids. Results differed majorly between protocols in the total number and subcellular compartment bias of identified proteins, which is particularly relevant for the reliable quantification of transporters and drug metabolizing enzymes. Using a model of metabolic dysfunction-associated steatotic liver disease, we furthermore show that critical disease pathways are robustly identified using a standardized high throughput-compatible workflow based on thermal lysis, even using only individual spheroids (1500 cells) as input. The results increase the applicability of proteomic profiling to phenotypic screens in organotypic microtissues and provide a scalable platform for deep phenotyping from limited biological material.
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| 2. |
- Xing, Chen, et al.
(författare)
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The choice of ultra-low attachment plates impacts primary human and primary canine hepatocyte spheroid formation, phenotypes, and function
- 2024
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Ingår i: Biotechnology Journal. - : John Wiley & Sons. - 1860-6768 .- 1860-7314. ; 19:2
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Tidskriftsartikel (refereegranskat)abstract
- Organotypic three-dimensional liver spheroid cultures in which hepatic cells retain their molecular phenotype and functionality have emerged as powerful tools for preclinical drug development. In recent years a multitude of culture systems have been developed; however, a thorough side-by-side benchmarking of the different methods is lacking. Here, we compared the performance of ten different 96- and 384-well microplate types to support spheroid formation and long-term culture. Specifically, we evaluated differences in spheroid formation kinetics, viability, functionality, expression patterns, and their utility for hepatotoxicity assessments using primary human hepatocytes (PHH) and primary canine hepatocytes (PCH). All 96-well plates enabled formation of PHH liver spheroids, albeit with differences between plates in spheroid size, geometry, and reproducibility. Performance of different 384-wells was less consistent. Only 6/10 microplates supported the formation of PCH aggregates. Interestingly, even if PCH aggregates in these six microplates were more loosely packed than PHH spheroids, they maintained their function and were compatible with long-term pharmacological and toxicological assays. Overall, Corning and Biofloat plates showed the best performance in the formation of both human and canine liver spheroids with highest viability, most physiologically relevant phenotypes, superior CYP activity and lowest coefficient of variation in toxicity assays. The presented data constitutes a valuable resource that demonstrates the impacts of current ultra-low attachment plates on liver spheroid metrics and can guide evidence-based plate selection. Combined, these results have important implications for the cross-comparison of different studies and can facilitate the standardization and reproducibility of three-dimensional liver culture experiments. We assessed the performance of ten different 384- and 96-well ultra-low attachment (ULA) microplates in facilitating three-dimensional spheroid culture of primary human hepatocytes (PHH) and primary canine hepatocytes (PCH) by examining effects on formation kinetics, spheroid morphology, long-term stability, functionality, expression signatures, and utility for hepatotoxicity assessment. Significant variations in experimental endpoints were observed between plates. These results can guide the optimization of spheroid experiments and contribute to the standardization of testing in three-dimensional organotypic liver cultures. image
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