Novel morphological multi-scale evaluation system for quality assessment of decellularized liver scaffolds

Abstract

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.

Identification of tissue sections from decellularized liver scaffolds for repopulation experiments

“…A pulsatile flow profile was applied, switching between 1 ml/min and 1.5 ml/min every 4 s, using the Harvard peristaltic pump p70 (Hugo-Sachs, Harvard Apparatus GmbH, Germany). The criteria for termination of the decellularization process were defined based on our experience from previous projects regarding the macroscopic appearance of the acellular liver [ 12 ]. We defined to stop the liver perfusion either when no remnant tissue or no macroscopic changes were seen for a period of more than 30 min.…”

“…In order to assess the influence of these factors in an easy to handle cell culture model, we and others propose the use of cell-free scaffold-sections [ 12 ] enabling high comparability of repopulation experiments.…”

Semantic Segmentation of Intralobular and Extralobular Tissue from Liver Scaffold H&E Images

“…The texture features were designed to describe the pixel intensity and the neighborhood texture. We started from our formerly designed method for scaffold texture segmentation [ 8 ] and extended the feature vector to better distinguish the differences between the “background” and the “intralobular” area. The flow-chart of the algorithm can be seen in Figure 2 .…”

“…Nevertheless, despite research efforts, the construction of liver tissue in vitro remains very challenging. The quality of decellularized scaffold is crucial for the initial cell-scaffold interaction [ 7 , 8 ], and thus determines the success of the cell repopulation process. However, the assessment of the scaffold quality prior to recellularization represents one of the remaining problems to be solved.…”

“…The first method for the quantitative evaluation of the structure quality with respect to particular liver scaffold features such as intralobular sinusoidal vessel structures was introduced in [ 8 ]. However, this method requires an initial user input thus it is observer dependent.…”