Human Organ Project and AST Transplantation and Immunology Research Network (TIRN) Research Grant: Defining Functional Standards in Bioreactor-Based Organ Engineering

30 million Americans suffer from end-stage renal disease, and the severe shortage of suitable donor kidneys causes thousands of patients in need of a transplant to die while undergoing extended dialysis while on the waiting list. By combining patient-specific cells and three-dimensional (3D) scaffolds within specially designed bioreactors, the discipline of de novo organ engineering could allow more patients to receive life-saving organ transplants by producing new, patient-customized transplantable grafts. While the de novo creation of transplantable human kidneys is a long-term goal, significant progress by our lab and others has been made in the derivation of cell-supporting kidney scaffolds through decellularization of non-transplantable donor kidneys. This strategy is performed by perfusing the renal vasculature with detergent solutions to extract the native cells, leaving behind an extracellular template upon which renal structures can regenerate. Our laboratory has developed perfusion-based bioreactors where we combine patient-derived cells and 3D extracellular matrix (ECM) scaffolds to regenerate renal structures in vitro, with the long-term goal of creating fully functional organs de novo. However, despite the progress in the field by our lab and others to develop ex vivo tissues for transplantation, the limiting factor is the need to non-invasively monitor cell growth, function, and number within 3D tissue developing within bioreactors. Therefore, the goal of this American Society of Transplantation-Human Organ Project Grant is to develop metrics and standards of cell growth and function within large-scale bioreactor systems to develop reference standards and calibration metrics for manufacturing in bioengineering to guide the future of de novo organ development. Our first aim for the present proposal is to refine and standardize a non-invasive cell quantification assay based on the metabolic reduction of resazurin, which is temporarily added to the culture media perfusate, to the highly fluorescent resorufin, by various populations of nephron-derived, vascular, and stromal cells grown within rodent kidney scaffolds. After standardizing the resazurin reduction assay, our second aim is to adapt this protocol to a normothermic human kidney preservation system. We will use the Functional Circulation Model 30 system to repopulate decellularized porcine or human kidneys with human renal tubule-derived epithelial cells. Results obtained from the resazurin reduction assay will be correlated with existing clinical prognostic indicators, including renal vascular resistance (based on pressure) and secreted biomarkers (e.g. kidney injury molecule-1) to create new standards for monitoring cellular viability, proliferation, renal function within partially regenerated human kidney scaffolds.