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（由申请人提供）：长期保存作为含有活细胞的再生医学产品的使能技术是至关重要的。然而，虽然冷冻保存对大多数细胞都很有效，但有些细胞更敏感，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因使用冷冻的传统冷冻保存方案形成的冰而受损。玻璃体冷冻保存是另一种冷冻保存策略，它使组织像玻璃一样稳定，没有冰结晶。然而，如果在玻璃化所需的高浓度冷冻保护剂的添加和去除过程中没有正确管理样品，则存在细胞毒性的风险。通过降低低温保护剂浓度来避免高浓度的低温保护剂配方，同时减少或抑制冰的形成，可以通过模仿某些昆虫在零度以下生存所采用的策略来实现。这些昆虫将冷冻保护剂含量（甘油）的增加与寒冷环境条件的开始结合起来，产生几种抗冻肽（afp），以在低至-80℃的温度下生存。这项可行性研究是为了确定使用这些昆虫afp来减少或抑制在常规条件下不能很好地冷冻保存的细胞的冰形成的潜在好处。这些afp控制细胞内外冰的能力将被研究。建立一种低温保存后产生bbbb75 %存活率的方案，然后优化并应用于第二阶段的组织。