*Purpose: In transplantation, the endothelial lining is the first barrier between the donor organ and recipient immune system. Damaged endothelium exposes extracellular matrix (ECM) molecules that can aggravate inflammation and cause graft rejection. Endothelial repair strategies may improve transplant outcomes. Here we prove that re-endothelialization of acellular blood vessels using kidney-vein endothelial cells (EC) generates a functional endothelium with vascular barrier function/innate immune conduit function.

*Methods: Human common iliac veins (CIV) (n=19) from deceased healthy donors were decellularized by submersion in Triton X-100 (4%), ammonia (1%) and DNase. Efficiency of decellularization was checked by residual DNA content analysis and histology. Decellularized CIV were subsequently repopulated with human umbilical vein endothelial cells (HUVEC) or patient derived kidney-vein EC. The re-endothelialized veins were analysed using confocal microscopy for EC confluency. Functionality of the EC barrier was analyzed using trans-endothelial electrical resistance (TEER), dextran permeability and nitric oxide production (eNOS). The innate immune barrier function was assessed by co-culture with THP-1 monocytic cells (5:1 ratio) in a house-built transmigration system.

*Results: The CIV were fully decellularized, demonstrated by the complete removal of cellular components, and the removal of dsDNA (before: 83.8±29.0 , after:13.0±6.5 ng/mg). Histological integrity was preserved, as well as ECM polysaccharides. Confocal microscopy showed the formation of a confluent monolayer of cells as soon as 24 hours after seeding. After 28 days of culture repopulated CIV scaffolds remained confluent. At day 10, the constructs had TEER measurements above background of 15.1±12.2 Ω·cm² (n=4); reduced dextran permeability compared to decellularized CIV; and showed eNOS activity. These results indicated the restoration of a functional EC barrier. The innate immune barrier function was demonstrated by THP-1 cell adhesion and transmigration through the EC monolayer. THP-1 differentiation into M1 inflammatory macrophages and M2 anti-inflammatory macrophages was confirmed via flow cytometry and immunohistochemistry with representative markers.

*Conclusions: We developed a procedure to efficiently decellularize human CIV and generated functional and long-term stable re-endothelialized veins using patient derived kidney-vein EC. This provides a new model to study re-endothelialization in-vitro.

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