Background: Since autologous veins are unavailable when needed in more than 20% of cases in vascular surgery, the production of personalized biological vascular grafts for implantation has become crucial. Surface modification of decellularized xenogeneic grafts with vascular cells to achieve physiological luminal coverage and eventually thromboresistance is an important prerequisite for implantation. However, ex vivo thrombogenicity testing remains a neglected area in the field of tissue engineering of vascular grafts due to a multifold of reasons. Methods: After seeding decellularized bovine carotid arteries with human endothelial progenitor cells and umbilical cord-derived mesenchymal stem cells, luminal endothelial cell coverage (LECC) was correlated with glucose and lactate levels on the cell supernatant. Then a closed loop whole blood perfusion system was designed. Recellularized grafts with a LECC > 50% and decellularized vascular grafts were perfused with human whole blood for 2 h. Hemolysis and complete blood count evaluation was performed on an hourly basis, followed by histological and immunohistochemical analysis. Results: While whole blood perfusion of decellularized grafts significantly reduced platelet counts, platelet depletion from blood resulting from binding to re-endothelialized grafts was insignificant (p = 0.7284). Moreover, macroscopic evaluation revealed thrombus formation only in the lumen of unseeded grafts and histological characterization revealed lack of CD41 positive platelets in recellularized grafts, thus confirming their thromboresistance. Conclusion: In the present study we were able to demonstrate the effect of surface modification of vascular grafts in their thromboresistance in an ex vivo whole blood perfusion system. To our knowledge, this is the first study to expose engineered vascular grafts to human whole blood, recirculating at high flow rates, immediately after seeding.

Primary human hepatocytes are widely used for basic research, pharmaceutical testing, and therapeutic concepts in regenerative medicine. Human hepatocytes can be isolated from resected liver tissue. Preoperative portal vein embolization (PVE) is increasingly used to decrease the risk of delayed postoperative liver regeneration by induction of selective hypertrophy of the future remnant liver tissue. The aim of this study was to investigate the effect of PVE on the outcome of hepatocyte isolation. Primary human hepatocytes were isolated from liver tissue obtained from partial hepatectomies (n=190) using the two-step collagenase perfusion technique followed by Percoll purification. Of these hepatectomies, 27 isolations (14.2%) were performed using liver tissue obtained from patients undergoing PVE prior to surgery. All isolations were characterized using parameters that had been described in the literature as relevant for the outcome of hepatocyte isolation. The isolation outcomes of the PVE and the non-PVE groups were then compared before and after Percoll purification. Metabolic parameters (transaminases, urea, albumin, and vascular endothelial growth factor secretion) were measured in the supernatant of cultured hepatocytes over a period of 6 days (PVE: n=4, non-PVE: n=3). The PVE and non-PVE groups were similar in regard to donor parameters (sex, age, indication for surgery), isolation parameters (liver weight, cold ischemic time), and the quality of the liver tissue. The mean initial viable cell yield did not differ between the PVE and non-PVE groups (10.16±2.03x106 cells/g vs. 9.70±0.73 x106 cells/g, p=0.499). The initial viability was slightly better in the PVE-group (77.8 ±2.03% vs. 74.4 ±1.06%). The mean viable cell yield (p=0.819) and the mean viability (p=0.141) after Percoll purification did not differ between the groups. PVE had no effect on enzyme leakage and metabolic activity of cultured hepatocytes. Although PVE leads to drastic metabolic alterations and changes in hepatic blood flow, embolized liver tissue is a suitable source for the isolation of primary human hepatocytes and is equivalent to untreated liver tissue in regard to cell yield and viability.

Purpose: Artificial organs might serve as alternative solutions for whole organ transplantation. Decellularization of a liver provides a non-immunogenic matrix with the advantage of three afferent systems, the portal vein, the hepatic artery and the bile duct. This study aims to evaluate the recellularization of rat livers with islets of Langerhans via the bile duct and the portal vein for the comparison of different perfusion routes. Methods: Rat livers were decellularized in a pressure-controlled perfusion manner and repopulated with intact isolated islets of Langerhans via either the portal vein or the bile duct. Results: Repopulation via the portal vein showed islet clusters stuck within the vascular system demonstrated by ellipsoid borders of thick reticular tissue around the islet cluster in Azan staining. After recellularization via the bile duct, islets were distributed close to the vessels within the parenchymal space and without a surrounding reticular layer. Large clusters of islets had a diameter of up to 1000 µm without clear shapes. Conclusion: We demonstrated the bile duct to be superior to the portal vein for repopulation of a decellularized rat liver with islets of Langerhans. This technique may serve as a bioengineering platform to generate an implantable and functional endocrine neo-pancreas and provide scaffolds with the anatomic benefit of three afferent systems to facilitate co-population of cells.

Vascular surgery faces a critical demand for novel vascular grafts that are biocompatible and thromboresistant. This urgency particularly applies to bypass operations involving small caliber vessels. In the realm of tissue engineering, the development of fully vascularized organs holds great promise as a solution to organ shortage for transplantation. To achieve this, it is imperative to (re-)construct a biocompatible and non-thrombogenic vascular network within these organs. In this systematic review, we identify, classify and discuss basic principles and methods used to perform in vitro/ex vivo dynamic thrombogenicity testing of perfusable tissue engineered organs and tissues. We conducted a pre-registered systematic review of studies published in the last 23 years according to PRISMA-P Guidelines, comprising a systematic data extraction, in-depth analysis and risk of bias assessment of 116 included studies. We identified shaking (n=28), flow loop (n=17), ex vivo (arterio-venous shunt, n=33) and dynamic in vitro models (n=38) as main approaches for thrombogenicity assessment. This comprehensive review unveils a prevalent lack of standardization and serves as a valuable guide in the design of standardized experimental setups.