Feasibility of expanding ischemia time for hearts destined for transplantation

延长移植心脏缺血时间的可行性

• 批准号: 10082625
• 负责人: Kelvin G.M. Brockbank
• 金额: $ 38.6万
• 依托单位: TISSUE TESTING TECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-23 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10082625
• 关键词: Air; AlamarBlue; Amino Acids; Animal Model; Animals; Anions; Antioxidants; Apoptosis; Apoptosis Inhibitor; Biological Assay; Blood; Blood Vessels; Body Fluids; Capital; Cardiac; Cardiac Myocytes; Cell Line; 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; Family suidae; Formulation; Gluconates; Gold; Grant; Heart; Heart Transplantation; Heterotopic Transplantation; Histopathology; Hour; Human; Hydrogen Sulfide; Ice; In Vitro; Inbred BALB C Mice; Intracellular Space; Ions; Ischemia; Isotonic Solutions; Lead; Letters; Lipids; Liver; Lobe; Mammals; Metabolic; Metabolism; Methodology; Methods; Molecular; Mus; Organ; Organ Donor; Organ Procurements; Organ Transplantation; Osmolalities; Outcome; Patients; Phase; Physiological; Plasma; Post-Translational Protein Processing; Postoperative Period; Potassium; Production; Proteomics; Pump; Reagent; Research; Safety; Saline; Ships; Small Business Innovation Research Grant; Sodium; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stains; Swelling; Technology; Testing; Time; Tissue imaging; Tissues; Transplantation; Trypan Blue; United Network for Organ Sharing; United States; Universities; Waiting Lists; Water; Work; World Health Organization; base; clinical practice; design; efficacy evaluation; experience; extracellular; glucose analog; heart function; heart preservation; improved; in vitro Assay; in vivo; inflammatory marker; innovation; macromolecule; meetings; metabolic rate; natural hypothermia; oxidation; post-transplant; preservation; pressure; scale up; screening; stress tolerance; tissue culture; transplant centers; transplant model; web site

In collaboration with Prof. Brandacher at Johns Hopkins University we plan to improve the currently most used clinical method of heart 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 a human cardiac myocyte cell line to select optimal reagent concentrations in vitro and a heterotopic heart 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. MALDI tissue imaging and proteomics will be performed on selected control and best performing hearts to provide molecular mapping information on metabolites, lipids, enzymatic products, and post translational modifications in heart sections. The in vivo studies will evaluate the best formulations from the in vitro studies over 7 days of hypothermic storage using a syngeneic BALB/C mouse, heterotopic transplantation model. Controls hearts will be preserved in high K+ Unisol™ (UHK) and HTK. We anticipate that the supplemented formulation will maintain viability and function of hearts for at least 36 hours, a considerable improvement over all current practice methods. Based upon preliminary data with blood vessel preservation, heart storage times of 6-7 days may be obtained. 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 heart storage in clinical practice that is relatively inexpensive and easy to ship by air. Demonstration of ≥36 hours of hypothermic storage with retention of heart function 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 heart 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常用的 心脏保存的临床方法，即 0 至 +4°C 的低温 我们将在此基础上进行。 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，恢复生理功能后功能正常 我们还表明，Unisol™ 在 -7° 条件下均可维持血管功能至少 6 天。 C 和 +4°C 以及在 Unisol™ 中 +4°C 下储存 18 小时的小鼠心脏的恢复速度明显加快 心脏功能优于金标准低温心脏保存液 Celsior (HTK) 中保存的心脏。 受到这些结果的鼓舞，我们建议对补充有针对氧化的试剂的 Unisol 进行评估， 细胞凋亡（应激耐受力的增强剂）和代谢（代谢率抑制），使用 人心肌细胞系体外最佳选择试剂浓度和异位心脏 移植模型来评估体外研究中开发的最佳补充剂配方。 体外检测将采用 alamarBlue，但结果将使用包括台盼在内的替代检测进行检查 如果代谢活性显着损失，将评估蓝色、活/死染色和细胞凋亡。 恢复生理培养条件后 1 至 2 天进行组织成像和蛋白质组学观察。 在选定的对照上进行并执行最佳心脏以提供分子图谱信息 心脏切片中的代谢物、脂质、酶产物和翻译后修饰。 研究将使用体外研究评估 7 天低温储存的最佳配方 同基因 BALB/C 小鼠，异位移植模型将保存在高 K+ 中。 我们预计补充配方将保持活力和活力。 心脏功能至少 36 小时，比目前所有的练习方法都有很大的进步。 根据血管保存的初步数据，可以获得6-7天的心脏保存时间。 创新不仅会增加存储时间，还将提供更紧密匹配的机会 此外，我们正在改进最尝试和最有效的方法。 临床实践中低温心脏储存的真正方法，相对便宜且简单 空运时需证明 ≥36 小时的低温储存并保留心脏功能。 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类心脏评估。