Chronic kidney disease (CKD) is a global health problem; the growing gap between the number of patients awaiting transplant and organs actually transplanted highlights the need for new treatments to restore renal function. Regenerative medicine is a promising approach from which treatments for organ-level disorders (e.g., neurogenic bladder) have emerged and translated to clinics. Regenerative templates, composed of biodegradable material and autologous cells, isolated and expanded ex vivo, stimulate native-like organ tissue regeneration after implantation. A critical step for extending this strategy from bladder to kidney is the ability to isolate, characterize, and expand functional renal cells with therapeutic potential from diseased tissue. In this study, we developed methods that yield distinct subpopulations of primary kidney cells that are compatible with process development and scale-up. These methods were translated to rodent, large mammal, and human kidneys, and then to rodent and human tissues with advanced CKD. Comparative in vitro studies demonstrated that phenotype and key functional attributes were retained consistently in ex vivo cultures regardless of species or disease state, suggesting that autologous sourcing of cells that contribute to in situ kidney regeneration after injury is feasible, even with biopsies from patients with advanced CKD.