Extracellular matrix- and pluripotent stem cell-based tissue engineering of the kidney

Complex, three-dimensional (3D) organ models are novel biotechnological tools for research on regeneration and disease mechanisms as well as drug development. An organ model of the kidney is urgently needed to break through decades of stagnation in the development of new treatment methods for patients with chronic kidney disease and to reduce the high failure rate of drugs in clinical tests due to nephrotoxic effects. The aim of this thesis was therefore the development of a human 3D kidney model. A functional kidney model should emulate the architecture and cell types of the kidney, as well as the mechanical properties and composition of the extracellular matrix. In order to emulate the function of the kidney, the model must be perfused. Therefore, a scaffold-based tissue engineering approach was chosen on the basis of decellularized whole rat kidneys, which should be recellularized with human renal precursor cells and endothelial cells differentiated from induced pluripotent stem cells. This approach required the development of a perfusion bioreactor and control software, which enabled the de- and recellularization of the kidneys as well as the subsequent in vitro cultivation. Decellularization is the process of removing all cells of an organ while preserving the extracellular matrix (ECM) in its native architecture and composition. The influence of different detergents and temperatures was analyzed, and the results showed that for the decellularization of tissue pieces immersion in the mild ionic detergent sodium deoxycholate (SDC) at 4 °C is optimal. However, decellularization of whole kidneys by perfusion required the strong ionic detergent sodium dodecyl sulfate (SDS). To improve the quality of the decellularized ECM it is beneficial to minimize the SDS concentration and application time as well as the temperature. All results were evaluated objectively and standardized by applying a point-based scoring system, which was also developed in the course of the thesis. Next, an efficient recellularization strategy had to be identified. The tested strategies included cell seeding through the renal artery with or without high pressure, seeding through the ureter with or without vacuum and direct injection into the parenchyma with a syringe. The vascular tree of the decellularized kidney was successfully recellularized with endothelial cells seeded via the renal artery. However, recellularization of the parenchyma with renal progenitor cells resulted in low seeding efficiencies in all applied seeding approaches. A maximum of 1% of the parenchyma could be repopulated by recellularization. In addition, the recellularization caused damage to the scaffold architecture and the arrangement of the cells did not correspond to the physiological renal structures. In parallel, the influence of mechanical and biochemical properties of the ECM on the maturation of renal progenitor cells was investigated. The cells were cultivated on surfaces of different stiffnesses and ECM coatings. With increasing stiffness, the renal progenitor cells increasingly matured into renal tubular epithelial cells, whereas podocyte maturation behaved inversely. In addition, the analysis revealed that the ECM protein laminin, in contrast to collagen IV, promotes the maturation of renal progenitor cells into renal tubular epithelial cells, although no difference was detected between the laminin isoforms 511 and 521. Although no kidney model could be generated with the investigated methods, the technical developments and the findings of this thesis mark a further step on the way to a human 3D kidney model.

Author dc:creator

• Fischer, Iris

Advisors dc:contributor.advisor

• Kurtz, Andreas
• Stachelscheid, Harald
• Reinke, Petra

Licence dc:rights.uri

• https://creativecommons.org/licenses/by/4.0/

Language dc:language.iso

en

Identifier URI

http://dx.doi.org/10.14279/depositonce-10529

OAI identifier oai:identifier

oai:depositonce.tu-berlin.de:11303/11641