In collaboration with Prof. Brandacher at Johns Hopkins University we plan to improve upon the currently mostused clinical method of limb preservation, namely hypothermia at 0 to +4°C. We will build upon our basepreservation formulation, Unisolâ¢, that has been shown to preserved whole large animals below +10°C for 6-8h after total blood replacement with Unisol⢠with normal functions upon return to physiological conditions.We have also shown that Unisol⢠can maintain blood vessel function for at least 6 days at both -7°C and +4°Cand that mouse hearts stored at 4°C for 18 hours in Unisol⢠have a significantly faster return of heart functionthan hearts stored in the gold standard hypothermic heart preservation solution Celsior (HTK). Encouraged bythese results we propose evaluation of Unisol supplemented with reagents targeting oxidation, apoptosis(enhancers of stress tolerance) and metabolism (metabolic rate inhibition) in 2 specific aims using humanskeletal myoblasts and vascular endothelial cells to select optimal reagent concentrations in vitro and a ratforelimb transplant model to evaluate the best supplement formulations developed in the in vitro studies. Thelead in vitro assay will be alamarBlue, however outcomes will be checked using alternative assays includingtrypan blue, live/dead stain and MTT assay. Apoptosis will be evaluated if significant losses of metabolicactivity are observed 1 to 2 days after return to physiologic culture conditions. The in vivo studies will evaluatethe best formulations from the in vitro studies over 3 days of hypothermic storage by transplantation model.Controls limbs will be preserved in Unisol⢠and HTK. We anticipate that the supplemented formulation willmaintain viability and function of limbs for up to 36 hours, a considerable improvement over all current practicemethods. This innovation will not only increase storage time, it will also provide the opportunity for more closelymatched recipients and potentially induction of tolerance. Furthermore, we are improving on the most tried andtrue method for hypothermic limb storage in clinical practice that is relatively inexpensive and easy to ship byair. Demonstration of â¥24 hours of hypothermic storage with >70% retention of limb functions will beconsidered to be a successful demonstration of feasibility for progression to a Phase II SBIR proposal forfurther evaluation in large animal models and ex vivo human limb evaluation post-preservation.
Public Health Relevance Statement: This proposal is focused on expanding the cold ischemia tolerance time for limbs so that more limbs will be
available for transplantation using a hypothermic preservation strategy that builds upon currently employed
clinical practices. Very few limb transplants are performed due in part to difficulties getting the limbs without
significant ischemia-induced damage. Many of the unused potential limb donations could have been employed
for transplantation if more time was available from the moment of organ procurement to transplant. The true
need for limb transplantation in the United States has been estimated to be several thousand upper extremity
limb recipients annually.
Project Terms: Air ; Amino Acids ; aminoacid ; Animals ; Anions ; Antioxidants ; anti-oxidant ; Biological Assay ; Assay ; Bioassay ; Biologic Assays ; Blood ; Blood Reticuloendothelial System ; Blood Vessels ; vascular ; Body Fluids ; Capital ; Cell membrane ; Cytoplasmic Membrane ; Plasma Membrane ; plasmalemma ; Cell Survival ; Cell Viability ; Cells ; Cell Body ; Charge ; Complement ; Complement Proteins ; Cryopreservation ; Cryofixation ; cold preservation ; cold storage ; Culture Media ; growth media ; Environment ; Equilibrium ; balance ; balance function ; Extracellular Space ; Intercellular Space ; Limb structure ; Extremities ; Limbs ; Non-Trunk ; Face ; faces ; facial ; Forelimb ; Genitalia ; Genital Organs ; Gluconates ; gluconic acid ; Gold ; Grant ; Cyclic GMP ; Guanosine Cyclic Monophosphate ; cGMP ; Heart ; Heart Transplantation ; Cardiac Transplantation ; Heart Grafting ; cardiac graft ; cardiovascular transplantation ; heart transplant ; Human ; Modern Man ; Hydrogen Sulfide ; natural hypothermia ; Hypothermia ; Ice ; In Vitro ; Ions ; Ischemia ; Isotonic Solutions ; Larynx ; Laryngeal ; Larynx Head and Neck ; voice box ; Lead ; Pb element ; heavy metal Pb ; heavy metal lead ; Mammals ; Mammalia ; Metabolism ; Intermediary Metabolism ; Metabolic Processes ; Methods ; Methodology ; Mus ; Mice ; Mice Mammals ; Murine ; Organ Preservation ; Organ Procurements ; oxidation ; Plasma ; Blood Plasma ; Plasma Serum ; Reticuloendothelial System, Serum, Plasma ; Postoperative Period ; Post-Operative ; Postoperative ; Potassium ; K element ; pressure ; Production ; Rattus ; Common Rat Strains ; Rat ; Rats Mammals ; Reagent ; Research ; Safety ; Saline ; Saline Solution ; Ships ; Sodium ; Na element ; Stains ; Staining method ; Swelling ; Family suidae ; Pigs ; Suidae ; Swine ; porcine ; suid ; Technology ; Testing ; Time ; tissue culture ; Tissues ; Body Tissues ; Trachea ; Trachea Proper ; windpipe ; Transplantation ; transplant ; Trypan Blue ; Benzamine Blue ; United States ; Universities ; Water ; Hydrogen Oxide ; Work ; Enhancers ; Osmolalities ; Apoptosis ; Apoptosis Pathway ; Programmed Cell Death ; base ; macromolecule ; Organ ; Pump ; improved ; Clinical ; Phase ; Physiological ; Physiologic ; Evaluation ; heart function ; cardiac function ; function of the heart ; Skeletal Muscle ; Voluntary Muscle ; AlamarBlue ; Alamar Blue ; Collaborations ; Letters ; Metabolic ; Hour ; stress tolerance ; extracellular ; Cl- element ; Chloride Ion ; experience ; glucose analog ; cardiac preservation ; heart preservation ; limb transplantation ; Embryonic Muscle Cells ; Precursor Muscle Cells ; Myoblasts ; Animal Models and Related Studies ; model of animal ; model organism ; Animal Model ; Histopathology ; Intracellular Space ; abdominal wall ; Skeletal Myoblasts ; Membrum superius ; Upper Limb ; Upper Extremity ; Vascular Endothelial Cell ; Apoptosis Inhibitor Gene ; Apoptosis Inhibitor ; Data ; in vitro Assay ; in vivo ; Small Business Innovation Research Grant ; SBIR ; Small Business Innovation Research ; Cardiac ; urinary ; Development ; developmental ; design ; designing ; efficacy evaluation ; efficacy analysis ; efficacy assessment ; efficacy examination ; evaluate efficacy ; examine efficacy ; Outcome ; scale up ; Consumption ; innovation ; innovate ; innovative ; inflammatory marker ; inflammation marker ; allotransplant ; allotransplantation ; clinical practice ; screening ; metabolic rate ; Formulation ; preservation ; transplant model ; post-transplant ; post-transplantation ; posttransplant ; posttransplantation ; Genital ;
