"Introduction: The lack of functional vascular networks is one of the most important hurdles in tissue engineering, limiting the obtainment of fully functional organs substitutes. Vascularization promotes efficient nutrients and oxygen supply, representing a key factor for in vivo application . Endothelial cells not only act as the structural building blocks of vascular endothelium, but they also fulfil key functional tasks by interacting with parenchymal cells in a complex cellular interplay. In the context of liver bioengineering, recent works described enhanced hepatic maturation when combining endothelial cells with liver organoids [2,3]. ‘Reset’ vascular endothelial cells (R-VECs) have recently been identified as a promising candidate for graft re-endothelialization by demonstrating good adaptability and efficient colonization capacity in decellularized scaffolds . The present work aims to combine R-VECs endothelial cells and human fetal liver organoids in decellularized ECM scaffold environment, to recapitulate the endothelial – parenchymal cells interplay.
Methodology: Human fetal liver organoids (post conception week, PCW = 5) were generated in Matrigel® 3D culture, after previous cells isolation via tissue disaggregation, and expanded in hepatic organoid expansion medium . R-VECs were cultured on conventional tissue culture flaks supplied by endothelial cells growth medium (EGM2) medium. Mouse livers were cannulated via the portal vein and decellularized via already established detergent-enzymatic treatment . 18 M cells were obtained from enzyme mediated organoid dissociation and were further seeded in decellularized scaffolds via portal vein injection. Repopulated livers were cultured in a custom-designed bioreactor in dynamic conditions provided by a peristaltic pump (flowrate = 3 ml/min) in hepatic organoid expansion medium for 7 days. Then, the same number of R-VECs were seeded with the same seeding technique. Repopulated scaffolds were cultured in the bioreactor with hepatic organoid expansion medium supplied with Oncostatin-M and Dexamethasone , and EGM2 in a 1:1 volume ratio. Histological and immunohistochemistry analysis were performed to study scaffold repopulation and the expression of mature hepatic and endothelial markers. qRT-PCR analysis was performed focusing on hepatic maturation markers such as cytochrome 3A4,1A2 (CYP3A4, CYP1A2), hepatocyte nuclear factor 4 alpha (HNF4α) and alpha fetoprotein (AFP).
Results: Mouse livers were successfully decellularized as highlighted by complete translucent appearance. H&E analysis showed efficient scaffold repopulation after 14 - days dynamic bioreactor culture. Immunofluorescence staining revealed the presence of hepatic maturation markers (HNF4α, human albumin and Alpha1-anti-trypsin) in co-presence of endothelial markers (CD31, Von Willebrand factor), highlighting epithelial-endothelial cell interaction and re-arrangement. qRT-PCR results showed enhanced expression of HNF4α, CYP3A4 and CYP1A2 in the 3D dynamic culture compared to the static in vitro control. Accordingly, lower AFP expression was also evidenced with respect to control in vitro culture.
Conclusion: Endothelial cells are involved in key structural and functional tasks that have a pivotal role in building up of functional organ substitutes. In the present work, human fetal liver organoids demonstrated increased phenotypic maturation when cocultured with R-VECs in a 3D organotypic environment. Endothelial cells demonstrated to be key players for the achievement of functional hepatic tissue, boosting fetal stage hepatocytes towards a more mature phenotype."