ABSTRACT: The broad aim of this program targets the banking of kidneys for transplantation. We propose to develop a nature inspired, holistic and non-toxic solution to dramatically extend preservation times during transplantation of human and xeno kidneys. Our approach is based on the best strategies employed by freeze- tolerant and hibernating animals in nature augmented with complementary strategies developed using recent scientific understanding and bioengineering principles. Importantly, our approach does not seek to solve all the problems needed for vitrification or classical cryopreservation, but rather be the first to develop organ preservation in a controlled, partially frozen or ice-free equilibrium state using high subzero temperatures (ranging from -5 to -30 °C) combined with metabolic depression. These are temperatures and strategies used in nature by species able to survive months in a state of ‘suspended animation,’ with the whole animal, including every single organ being “banked” without injury. We have conceived an integrated approach in which we will develop a new stasis cocktail optimized for the critical phases of protection (prior to storage), preservation (during storage) and revival-resuscitation (after storage). A critical aspect for translation of this technology to complex organs will be designing and optimizing a stasis cocktail which will serve the heterogeneous cell types of the kidney together with well-developed model systems for renal transplant. Across three specific aims, in this first phase, we use representative renal cell types - Human Renal Proximal Tubular Epithelial Cells (HRPTEpiC) and Human Renal Glomerular Endothelial Cells (HRGEC) - as a simplified in vitro kidney model. The objective is to develop a (1) nature-inspired, bioengineering augmented non-toxic cryostasis cocktail that seeks to accomplish a broader set of goals than traditional cryoprotectants and (2) pre-conditioning, cooling, and rewarming protocol for high subzero cryopreservation in the presence of limited, controlled ice or total absence of ice in a thermodynamically stable state. Specifically, the study is designed to contrast the efficacy of a partial freezing strategy with an ice-free approach, both of which are employed in nature. We will combine components that enable: (a) resistance to deleterious changes in cell volume and prevention of intracellular ice formation through the use of low molecular weight compounds,(b) the active suppression of metabolic rate beyond what is possible through passive temperature effects alone (through the application of metabolic rate inhibitors), and (c) the enhancement of stress tolerance and reduction of cryoinjury through the application of antioxidants and pro- survival compounds. The best conditions will then be evaluated using a whole kidney model in Phase I, and ultimately kidney transplant in Phase II. This specific proposal is an important standalone project that also should lead to solutions for preservation of natural and engineered vital organs, as well as for traditional cell and tissue banking. Ultimately, we anticipate achieving up to 10-14 days banking using the best of these approaches which would be a game-changer for transplantation and allow time for induction of immune tolerance in the recipient.

Narrative The need for organ transplantation is continuing to grow steadily and official U.S. organ transplant waitlist statistics show that there are 5X the number of people on the waitlist than received a transplant last year (United Network of Organ Sharing (UNOS) data) and other estimates suggest that 35% of all annual U.S. deaths would be prevented by organ transplantation. At the same time, many vital organs are left unused due to the short amount of time that organs can be kept alive for transport and of those used the risk of ischemic and other forms of damage still exists and can contribute to graft rejection. Short preservation times and risk of ischemia also constrain optimal donor-recipient matching and prevent the ultimate strategy of immune tolerance induction. Our technology promises to overcome these issues by markedly extending the preservation time enabling tissue-matched organs stored for sufficient time (weeks to months) to meet the challenges of global transportation and immune tolerance induction.