Controlling Intracellular Ice Formation with Antifreeze Proteins

用抗冻蛋白控制细胞内冰的形成

• 批准号: 7746586
• 负责人: LIA H CAMPBELL
• 金额: $ 20.31万
• 依托单位: CELL AND TISSUE SYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-03-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7746586
• 关键词: Antifreeze Proteins; Architecture; Arctic Regions; Biological Preservation; Cell Line; Cell Survival; Cell Therapy; Cells; Chemicals; Cryopreservation; Crystallization; Cultured Cells; Detection; Drug Formulations; Evaluation; Excision; Feasibility Studies; Fishes; Freezing; Glass; Glycerol; Goals; Growth; Ice; Insecta; Life; Methods; Organ; Organism; Patients; Peptides; Phase; Price; Process; Production; Propylene Glycols; Protocols documentation; Recombinants; Regenerative Medicine; Research; Residual state; Risk; Sampling; Screening procedure; Small Business Innovation Research Grant; Smooth Muscle Myocytes; Solutions; Sulfoxide; System; Technology; Temperature; Tissue Engineering; Tissues; Transplanted tissue; Viscosity; Work; base; carcinogenicity; cell injury; cytotoxic; cytotoxicity; extracellular; novel; public health relevance; research study; three dimensional structure

DESCRIPTION (provided by applicant): Long term preservation is crucial as an enabling technology for regenerative medicine products that contain living cells. However, while cryopreservation works well for most cells, some cells are more sensitive and hard to cryopreserve by freezing. Plus, the three dimensional structure and extracellular architecture of tissues is damaged by ice formation that results from more conventional cryopreservation protocols that use freezing. Vitreous cryopreservation is an alternative cryopreservation strategy that stabilizes the tissue as a glass, no ice crystallization. However, there is the risk of cytotoxicity if the sample is not managed correctly during both addition and removal of the high concentrations of cryoprotectants required for vitrification. Avoidance of high concentration cryoprotectant formulations by reduction of cryoprotectant concentrations while reducing or inhibiting ice formation may be possible by mimicking the strategy employed by certain insects to survive sub-zero temperatures. These insects combine increases in cryoprotectant content (glycerol) with the onset of cold environmental conditions with production of several antifreeze peptides (AFPs) to survive temperatures as low as -80¿C. This feasibility study is to determine the potential benefits of using these insect AFPs to reduce or inhibit ice formation for the cryopreservation of cells that do not cryopreserve well under conventional conditions. The ability of these AFPs to control ice both inside and outside the cell will be investigated. Establishment of a protocol that produces >75% viability after cryopreservation will then be optimized and applied to tissues in Phase II. PUBLIC HEALTH RELEVANCE: Long term storage technologies are necessary for newly developed cell-based therapies. Cryopreservation is the most obvious avenue for storage but ice formation in the cells and tissues can cause irreparable damage. The use of high concentrations of cryoprotectants would facilitate avoidance of ice but at the price of cytotoxicity. This proposal will develop preservation protocols using antifreeze proteins such that less cryoprotectants are required and ice formation is minimized or eliminated. This strategy would provide preservation methods for cell and tissue transplants that may eventually impact more than a 100 million US patients.

描述(申请人提供)：对于含有活细胞的再生医学产品来说，长期保存是一项至关重要的技术。然而，尽管冷冻保存对大多数细胞都很有效，但一些细胞更敏感，很难通过冷冻进行冷冻保存。此外，组织的三维结构和细胞外结构会受到冰层形成的破坏，这种冰层是由使用冷冻的更传统的冷冻保存方案造成的。玻璃体冷冻保存是一种替代的冷冻保存策略，它将组织稳定为玻璃，没有冰结晶。然而，如果在添加和移除玻璃化所需的高浓度冷冻保护剂期间没有正确地管理样品，则存在细胞毒性的风险。通过降低低温保护剂的浓度，同时减少或抑制结冰，可以通过模仿某些昆虫在零度以下生存的策略来避免高浓度的低温保护剂配方。这些昆虫结合了低温保护剂(甘油)含量的增加和寒冷环境条件的开始，以及几种抗冻肽(AFP)的产生，以在低至-80°C的温度下生存。这项可行性研究旨在确定使用这些昆虫AFP来减少或抑制冰形成的潜在好处，用于在常规条件下不能很好地低温保存的细胞的低温保存。这些AFP控制细胞内外结冰的能力将被调查。建立一种在冷冻保存后产生75%存活率的方案将被优化，并在第二阶段应用于组织。 公共卫生相关性：长期储存技术对于新开发的基于细胞的疗法是必要的。超低温保存是最明显的保存方法，但细胞和组织中的结冰可能会造成无法弥补的损害。使用高浓度的低温保护剂将有助于避免结冰，但代价是细胞毒性。这项提案将开发使用抗冻蛋白的保存方案，从而减少需要的冷冻保护剂，最大限度地减少或消除结冰。这一战略将为细胞和组织移植提供保存方法，最终可能影响超过1亿美国患者。