Extended limb preservation employing an optimization strategy for stabilization.

采用优化稳定策略来延长肢体保护。

• 批准号: 10257524
• 负责人: Kelvin G.M. Brockbank
• 金额: $ 33.95万
• 依托单位: TISSUE TESTING TECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10257524
• 关键词: Air; AlamarBlue; Amino Acids; Animal Model; Animals; Anions; Antioxidants; Apoptosis; Apoptosis Inhibitor; Biological Assay; Blood; Blood Vessels; Body Fluids; Capital; Cardiac; Cell Survival; Cell membrane; Cells; Charge; Chloride Ion; Clinical; Collaborations; Complement; Consumption; Cryopreservation; Culture Media; Cyclic GMP; Data; Development; Enhancers; Environment; Equilibrium; Evaluation; Extracellular Space; Face; Family suidae; Forelimb; Formulation; Genital; Genitalia; Gluconates; Gold; Grant; Heart; Heart Transplantation; Histopathology; Hour; Human; Hydrogen Sulfide; Ice; In Vitro; Intracellular Space; Ions; Ischemia; Isotonic Solutions; Larynx; Lead; Letters; Limb structure; Mammals; Metabolic; Metabolism; Methodology; Methods; Mus; Myoblasts; Organ; Organ Preservation; Organ Procurements; Osmolalities; Outcome; Phase; Physiological; Plasma; Postoperative Period; Potassium; Production; Pump; Rattus; Reagent; Research; Safety; Saline; Ships; Skeletal Muscle; Skeletal Myoblasts; Small Business Innovation Research Grant; Sodium; Stains; Swelling; Technology; Testing; Time; Tissues; Trachea; Transplantation; Trypan Blue; United States; Universities; Upper Extremity; Vascular Endothelial Cell; Water; Work; abdominal wall; allotransplant; base; clinical practice; design; efficacy evaluation; experience; extracellular; glucose analog; heart function; heart preservation; improved; in vitro Assay; in vivo; inflammatory marker; innovation; limb transplantation; macromolecule; metabolic rate; natural hypothermia; oxidation; post-transplant; preservation; pressure; scale up; screening; stress tolerance; tissue culture; transplant model; urinary

In collaboration with Prof. Brandacher at Johns Hopkins University we plan to improve upon the currently most used clinical method of limb preservation, namely hypothermia at 0 to +4°C. We will build upon our base preservation formulation, Unisol™, 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°C and that mouse hearts stored at 4°C for 18 hours in Unisol™ have a significantly faster return of heart function than hearts stored in the gold standard hypothermic heart preservation solution Celsior (HTK). Encouraged by these 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 human skeletal myoblasts and vascular endothelial cells to select optimal reagent concentrations in vitro and a rat forelimb transplant model to evaluate the best supplement formulations developed in the in vitro studies. The lead in vitro assay will be alamarBlue, however outcomes will be checked using alternative assays including trypan blue, live/dead stain and MTT assay. Apoptosis will be evaluated if significant losses of metabolic activity are observed 1 to 2 days after return to physiologic culture conditions. The in vivo studies will evaluate the 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 will maintain viability and function of limbs for up to 36 hours, a considerable improvement over all current practice methods. This innovation will not only increase storage time, it will also provide the opportunity for more closely matched recipients and potentially induction of tolerance. Furthermore, we are improving on the most tried and true method for hypothermic limb storage in clinical practice that is relatively inexpensive and easy to ship by air. Demonstration of ≥24 hours of hypothermic storage with >70% retention of limb functions will be considered to be a successful demonstration of feasibility for progression to a Phase II SBIR proposal for further evaluation in large animal models and ex vivo human limb evaluation post-preservation.

我们计划与约翰·霍普金斯大学的 Brandacher 教授合作，改进目前最先进的 采用临床保肢方法，即0至+4°C低温。我们将在我们的基础上再接再厉 保存配方 Unisol™，已被证明可以在 +10°C 以下保存整个大型动物 6- 用 Unisol™ 全血置换后 8 小时，恢复生理状态后功能正常。 我们还证明 Unisol™ 在 -7°C 和 +4°C 下均可维持血管功能至少 6 天 小鼠心脏在 Unisol™ 中于 4°C 保存 18 小时，心脏功能的恢复速度显着加快 与金标准低温心脏保存液 Celsior (HTK) 中保存的心脏相比。受到鼓励 根据这些结果，我们建议对补充有针对氧化、细胞凋亡的试剂的 Unisol 进行评估 （增强压力耐受力）和新陈代谢（代谢率抑制），使用人体来实现 2 个特定目标 骨骼肌成肌细胞和血管内皮细胞在体外和大鼠中选择最佳试剂浓度 前肢移植模型来评估体外研究中开发的最佳补充剂配方。这 主要体外检测将是 alamarBlue，但是将使用其他检测来检查结果，包括 台盼蓝、活/死染色和 MTT 测定。如果代谢显着损失，将评估细胞凋亡 返回生理培养条件后1至2天观察活性。体内研究将评估 通过移植模型进行 3 天低温储存的体外研究得出的最佳配方。 控制肢体将保存在 Unisol™ 和 HTK 中。我们预计补充配方将 维持肢体的活力和功能长达 36 小时，比目前的所有实践都有相当大的进步 方法。这项创新不仅会增加存储时间，还将提供更紧密的机会 匹配的接受者并可能诱导耐受。此外，我们正在改进最经过尝试和 临床实践中低温肢体储存的真正方法，相对便宜且易于运输 空气。证明 ≥24 小时的低温储存且肢体功能保留 >70% 被认为成功证明了进入第二阶段 SBIR 提案的可行性 大型动物模型的进一步评估和保存后的离体人体肢体评估。