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Decellularized scaffolds can serve as an excellent three-dimensional environment for cell repopulation. They maintain tissue-specific microarchitecture of extracellular matrix proteins with important spatial cues for cell adhesion, migration, growth, and differentiation. However, criteria for quality assessment of the three-dimensional structure of decellularized scaffolds are rather fragmented, usually study-specific, and mostly semi-quantitative. Thus, we aimed to develop a robust structural assessment system for decellularized porcine liver scaffolds. Five scaffolds of different quality were used to establish the new evaluation system. We combined conventional semi-quantitative scoring criteria with a quantitative scaffold evaluation based on automated image analysis. For the quantitation, we developed a specific open source software tool (ScaffAn) applying algorithms designed for texture analysis, segmentation, and skeletonization. ScaffAn calculates selected parameters characterizing structural features of porcine liver scaffolds such as the sinusoidal network. After evaluating individual scaffolds, the total scores predicted scaffold interaction with cells in terms of cell adhesion. Higher scores corresponded to higher numbers of cells attached to the scaffolds. Moreover, our analysis revealed that the conventional system could not identify fine differences between good quality scaffolds while the additional use of ScaffAn allowed discrimination. This led us to the conclusion that only using the combined score resulted in the best discrimination between different quality scaffolds. Overall, our newly defined evaluation system has the potential to select the liver scaffolds most suitable for recellularization, and can represent a step toward better success in liver tissue engineering. [ABSTRACT FROM AUTHOR]