Konstruktion eines Neo-Pankreas mittels Dezellularisierung und Rezellularisierung

"Die auf Langerhans-Inseln basierende Rezellularisierung einer dezellularisierten Rattenleber könnte die Grundlage für ein transplantables Neo-Pankreas darstellen. Das Ziel dieser Arbeit bestand in der Etablierung der erforderlichen Protokolle, der Evaluierung der geschaffenen Organstruktur sowie der Funktionsanalyse der Langerhans-Inseln ex vivo. Nach der flussgesteuerten Dezellularisierung von Rattenlebern wurden diese mit Endothelzellen und mesenchymalen Stromazellen der Ratte rebesiedelt, für 8 Tage in einer Perfusionskammer kultiviert und schließlich am 9. Tag mit Langerhans-Inseln der Ratte rebesiedelt. Die Integrität und Funktionalität der Re-Endothelialisierung wurde mittels histologischer Färbungen und der Perfusion mit FITC-Dextran validiert. Die Funktionalität der Langerhans-Inseln wurde an Tag 10 und 12 mit Hilfe eines Glukose-induzierten Insulinsekretionstests überprüft. Mittels Blutgasanalyse erhobene Parameter bestätigten die Stabilität der Perfusionskultur. Die histologische Auswertung zeigte, dass Endothelzellen innerhalb der intakten Gefäßstruktur ein Monolayer ausbildeten. Die elektronenmikroskopischen Aufnahmen bestätigten diese Ergebnisse. Die Langerhans-Inseln, die über den Gallengang infundiert wurden, konnten in der histologischen Auswertung nachgewiesen werden. Eine adäquate Insulinsekretion nach Glukosestimulation sowohl in der eintägigen als auch nach dreitägiger Kultur bestätigte die erhaltene Viabilität und Funktionalität. Diese Arbeit liefert somit den Nachweis für eine erfolgreiche Kultivierung von Langerhans-Inseln in einer dezellularisierten Rattenleber. Im Rahmen des Projekts implementierten wir die Re-Endothelialisierung des Gefäßsystems als notwendige Voraussetzung für Implantation und Vollblutperfusion. Diese Techniken können als Plattform zu Generierung eines implantierbaren und funktionalen, endokrinen Neo-Pankreas gesehen werden." (Übersetzung aus Everwien et al. Engineering an endothelialized, endocrine Neo-Pancreas: Evaluation of islet functionality in an ex vivo model, Acta Biomater. 117 (2020) 213–225. https://doi.org/10.1016/j.actbio.2020.09.022.).“Islet-based recellularization of decellularized, repurposed rat livers may form a transplantable Neo-Pancreas. The aim of this study is the establishment of the necessary protocols, the evaluation of the organ structure and the analysis of the islet functionality ex vivo. After perfusion-based decellularization of rat livers, matrices were repopulated with endothelial cells and mesenchymal stromal cells, incubated for 8 days in a perfusion chamber and finally repopulated on day 9 with intact rodent islets. Integrity and quality of re-endothelialization was assessed by histology and FITC-dextran perfusion assay. Functionality of the islets of Langerhans was determined on day 10 and day 12 via glucose stimulated insulin secretion. Blood gas analysis variables confirmed the stability of the perfusion cultivation. Histological staining showed that cells formed a monolayer inside the intact vascular structure. These findings were confirmed by electron microscopy. Islets infused via the bile duct could histologically be found in the parenchymal space. Adequate insulin secretion after glucose stimulation after 1-day and 3-day cultivation verified islet viability and functionality after the repopulation process. We provide the first proof-of-concept for the functionality of islets of Langerhans engrafted in a decellularized rat liver. Furthermore, a re-endothelialization step was implemented to provide implantability. This technique can serve as a bioengineered platform to generate implantable and functional endocrine Neo-Pancreases.” [1

In vitro recellularization of decellularized bovine carotid arteries using human endothelial colony forming cells

Background: Many patients suffering from peripheral arterial disease (PAD) are dependent on bypass surgery. However, in some patients no suitable replacements (i.e. autologous or prosthetic bypass grafts) are available. Advances have been made to develop autologous tissue engineered vascular grafts (TEVG) using endothelial colony forming cells (ECFC) obtained by peripheral blood draw in large animal trials. Clinical translation of this technique, however, still requires additional data for usability of isolated ECFC from high cardiovascular risk patients. Bovine carotid arteries (BCA) were decellularized using a combined SDS (sodium dodecyl sulfate) -free mechanical-osmotic-enzymatic-detergent approach to show the feasibility of xenogenous vessel decellularization. Decellularized BCA chips were seeded with human ECFC, isolated from a high cardiovascular risk patient group, suffering from diabetes, hypertension and/or chronic renal failure. ECFC were cultured alone or in coculture with rat or human mesenchymal stromal cells (rMSC/hMSC). Decellularized BCA chips were evaluated for biochemical, histological and mechanical properties. Successful isolation of ECFC and recellularization capabilities were analyzed by histology. Results: Decellularized BCA showed retained extracellular matrix (ECM) composition and mechanical properties upon cell removal. Isolation of ECFC from the intended target group was successfully performed (80% isolation efficiency). Isolated cells showed a typical ECFC-phenotype. Upon recellularization, co-seeding of patient-isolated ECFC with rMSC/hMSC and further incubation was successful for 14 (n = 9) and 23 (n = 5) days. Reendothelialization (rMSC) and partial reendothelialization (hMSC) was achieved. Seeded cells were CD31 and vWF positive, however, human cells were detectable for up to 14 days in xenogenic cell-culture only. Seeding of ECFC without rMSC was not successful. Conclusion: Using our refined decellularization process we generated easily obtainable TEVG with retained ECM- and mechanical quality, serving as a platform to develop small-diameter (< 6 mm) TEVG. ECFC isolation from the cardiovascular risk target group is possible and sufficient. Survival of diabetic ECFC appears to be highly dependent on perivascular support by rMSC/hMSC under static conditions. ECFC survival was limited to 14 days post seeding

Surface modification of decellularized bovine carotid arteries with human vascular cells significantly reduces their thrombogenicity

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