SBIR-STTR Award

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.

Public Health Relevance Statement:
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.

Project Terms:
Address; Animals; animation; Antioxidants; Apoptosis Inhibitor; Area; base; Biological Models; Biomedical Engineering; Biomedical Research; cell type; Cell Volumes; Cells; Cessation of life; combinatorial; Complex; Coupled; Cryopreservation; Data; data sharing; design; Development; Donor person; Endothelial Cells; Engineering; Epithelial Cells; Equilibrium; Evaluation; Freezing; glomerular endothelium; Glucose; Goals; Graft Rejection; Heart; Human; Ice; Immune Tolerance; In Vitro; inhibitor/antagonist; Injury; Ischemia; Kidney; kidney cell; Kidney Transplantation; Lead; Left; Length; Letters; Liver; Lobe; Metabolic; metabolic depression; metabolic rate; Metabolism; Miniature Swine; Modeling; Molecular Weight; Nature; novel strategies; Organ; Organ Model; Organ Preservation; Organ Transplantation; Oryctolagus cuniculus; Perfusion; Phase; Phospholipids; preconditioning; preservation; prevent; Prevention; programs; Protocols documentation; prototype; Quality of life; Rana; Rattus; Recovery; Reperfusion Injury; Resistance; Resuscitation; Rewarming; Risk; scale up; Squirrel; statistics; stress reduction; stress tolerance; stressor; success; System; Technology; Temperature; Testing; Thermodynamics; Time; Tissue Engineering; Tissue Preservation; Tissues; Translations; Transplant Recipients; Transplantation; Transportation; trauma care; Tubular formation; United Network for Organ Sharing; Validation; Waiting Lists; Wood material

The kidney transplant waitlist comprises 83% of the U.S. organ transplant waitlist. Yet, for every 5 patients who dieor become too sick for transplantation, 3 kidneys are discarded. Organ preservation quality is linked to graft lifespanwith kidneys having only 50% graft survival after 10 years. Extending preservation to a week, or longer, would enablenationwide (theoretically global) donor-recipient matching, allowing many good quality kidneys not transplanted today(e.g., extended criteria donor kidneys) to be offered to the patients who most need them. Kidney preservation timesof 7-9 days would enable translation of clinical immune tolerance induction, and avoidance of life-longimmunosuppression requirements, from contexts of living donor transplantation to deceased organ donation(80% of all transplants), improving the lives of transplant recipients, and saving the healthcare system ~ $100 milliondollars each year. Improved and extended preservation technology, with off-the-shelf availability, can enhancescreening for transmissible diseases/malignancies, allow assessment of organ quality, drug/gene therapies for graftfunction augmenting, and support xeno-transplantation research. Moreover, 3D slices from preserved kidneysunmatched for transplant, have wide applicability in drug safety/efficacy testing, and a large variety of research topics. Building on the project's Phase I feasibility success that demonstrated unprecedented (rabbit) kidneypreservation for up to 14 days, we here scale-up this new technology to develop a (i) nature-inspired and(ii) machine perfusion enabled, subzero solution to dramatically extend preservation times of human andporcine (xeno) kidneys. High subzero temperatures (-12 to -20°C) applied using an ultra-stable equilibriumapproach are combined with stress tolerance enhancement and metabolic suppression strategies. Optimizedmachine perfusion protocols, 3D combinatorial high throughput screening with precision cut kidney slices,advanced µCT imaging, whole human kidney, and porcine survival auto transplantation models are employed.Based upon the significant Phase I achievements and our team's organ preservation experience, we developtechnologies and strategies in Phase II for enhanced human kidney equilibrium ice-free high subzero bankingvia: 1) multi-temperature multi-step machine perfusion platform supportive of pre-conditioning protocols, for clinicalscale kidney effective and uniform permeation, at all renal compartmental levels, with (i) biocompatible cryostasiscocktails formulated on the basis of toxicity neutralization and mutual dilution principles, and (ii) effectiverevival/reconditioning protocols for enhanced reperfusion; 2) validation of (168-216h) 7-9 days preservation incomplete absence of ice with (iii) cross country shipping of stored kidneys, and clinical practices of ex vivo renalperfusion; 3) porcine model kidney orthotopic transplantation with long-term survival, with and without "anywherein the world" shipping, and 4) randomized physiologic ex vivo assessment trial of 7-9 days stored human kidneys. This Phase II sets the stage for logistical model of centralized centers of excellence for preservation andassessment, enabling vastly improved clinical outcomes and patients' access across the country, and world.

Public Health Relevance Statement:
NARRATIVE Every year, thousands of potentially transplantable kidneys are not used and for every 5 waitlist patients who die, or become too sick for transplantation, 3 kidneys are discarded. Since quality of preservation/organ is linked to graft lifespan, with kidneys having only 50% graft survival after 10 years, extending kidney preservation to 7-9 days would address organ shortage by (a) enabling clinical immune tolerance induction, (b) greatly expanding options for donor-recipient matching and equitable allocation, (c) enhancing screening for transmissible diseases and malignancies, (d) decreasing costs and enabling flexible scheduling of surgeries, and (e) allowing for organ quality assessment and reconditioning. To address these needs, we develop a nature-inspired and machine perfusion enabled, subzero solution to dramatically extend preservation times of human and porcine (xeno) kidneys to (168-216h) 7-9 days, with thevalidation towards clinical application being accomplished with renal orthotopic autotransplantation in a porcine model, without and with "anywhere in the world" shipping, and complementary randomized physiologic ex-vivo assessment trial of 7-9 days stored human kidneys.

Project Terms:
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