Introduction: 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. Preservation and restoration of the endothelial barrier function is thus crucial for the normal performance of the kidney vascular system after transplantation. Here we prove that re-endothelialization of acellular blood vessels using patient derived kidney-vein endothelial cells (EC) restores both the vascular barrier function and innate immune function of the endothelium.

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. Efficacy of the process was evaluated via histological analysis and quantification of DNA. The ECM protein makeup preservation was assessed via collagen and sGAG content. 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 (4 and 70 kDa) permeability and nitric oxide production (eNOS). The innate immune barrier function of recellularized scaffolds was assessed by co-culture with THP-1 monocytic cells (5:1 ratio) in a home 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 and collagen. 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 and cells expressed the proliferation marker Ki-67 and PECAM-1. 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 (2.3-fold for 4kDa and 4.7-fold for 70kDa; n=3); and showed higher nitrate and nitrite concentration compared to plastic cultures (n=4). These results indicated the restoration of a functional EC barrier. The innate immune barrier function was demonstrated by THP-1 cell adhesion (only on recellularized scaffolds) 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 (CD14, CD16, CD80 and CD163).

Conclusion: We used an in-vitro ECM blood vessel model to prove functionality of recellularized human vein tissue with patient-derived kidney vein EC. Repopulated scaffolds also showed immune innate function, paving the way to future translation into clinical practice.