Identification of tissue sections from decellularized liver scaffolds for repopulation experiments

Abstract

Background: Biological organ engineering is a novel experimental approach to generate functional liver grafts by decellularization and repopulation. Currently, healthy organs of small or large animals and human organs with preexisting liver diseases are used to optimize decellularization and repopulation. However, the effects of morphological changes on allo-and xenogeneic cell-scaffold interactions during repopulation procedure, e.g., using scaffold-sections, are unknown. We present a sequential morphological workflow to identify murine liver scaffold-sections with well-preserved microarchitecture. Methods: Native livers (CONT, n ¼ 9) and livers with experimentally induced pathologies (hepatics steatosis: STEA, n ¼ 7; hepatic fibrosis induced by bile duct ligation: BDL, n ¼ 9; nodular regenerative hyperplasia induced by 90% partial hepatectomy: PH, n ¼ 8) were decellularized using SDS and Triton X-100 to generate cell-free scaffolds. Scaffold-sections were assessed using a sequential morphological workflow consisting of macroscopic, microscopic and morphological evaluation: (1) The scaffold was evaluated by a macroscopic decellularization score. (2) Regions without visible tissue remnants were localized for sampling and histological processing. Subsequent microscopical examination served to identify tissue samples without cell remnants. (3) Only cell-free tissue sections were subjected to detailed liver-specific morphological assessment using a histological and immunohistochemical decellularization score. Results: Decellularization was feasible in 33 livers, which were subjected to the sequential morphological workflow. In 11 of 33 scaffolds we achieved a good macroscopic decellularization result (CONT:

Recent Advances in Liver Engineering With Decellularized Scaffold

“…Ionic, non-ionic, and zwitterionic detergents are commonly used for decellularization. The most extensively used ionic detergents are sodium dodecyl sulfate (SDS) and sodium deoxycholate (SDC), which solubilize cellular membranes and denature proteins ( Zhang et al, 2018 ; Willemse et al, 2020 ; Felgendreff et al, 2021 ). Triton X-100 is the most widely used non-ionic detergent and is capable of breaking lipid-lipid, lipid-protein, and DNA-protein interactions.…”

“…Philipp et al recently used macroscopic, microscopic, and morphological assessments to develop a morphological workflow for analyzing the microstructure of decellularized mouse liver scaffolds with mild histological damage (hepatic steatosis, hepatic fibrosis, and nodular regenerative hyperplasia) (details presented in Table 2 ). The results indicated that this procedure aided in evaluating the quality of decellularized scaffolds and in identifying the tissues with the best-preserved microstructure and matrix components; these results are also fundamental for future repopulation and transplantation trials ( Felgendreff et al, 2021 ).…”

The potential of organoids in toxicologic pathology: role of toxicologic pathologists in <i>in vitro</i> chemical hepatotoxicity assessment

“…This method involves liver decellularization by refluxing hepatocytes and non-hepatocytes with Triton-X and/or sodium dodecyl sulfate and using the remaining stromal tissue as a template to redistribute the cells to be cultured 42 . In this method, the remaining hepatocytes and DNA interfere with the analysis after redistribution; therefore, reflux and validation methods were modified 43 , 44 . Using this method, several researchers have investigated the differentiation of bone marrow-derived mesenchymal cells into hepatocytes 45 .…”

Porta-caval fibrous connections — the lesser-known structure of intrahepatic connective-tissue framework: A unified view of liver extracellular matrix

“…The emergence of endoscopic anatomic liver resections strengthened the need to specify the anatomy and interrelationship of the connective-tissue structures within the liver[ 8 - 11 ]. Additionally, the prospects for the use of human and animal liver matrices as scaffolds for the creation of bioartificial livers (thanks to the development of stem cells and bioengineering technologies)[ 11 - 14 ] also contribute to the resurgence of interests in the hepatic connective-tissue structures.…”