Controlling Intracellular Ice Formation with Antifreeze Proteins

描述（由申请人提供）：长期保存是包含活细胞的再生医学产品的关键技术。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，难以通过冷冻进行冷冻保存。另外，组织的三维结构和细胞外结构被冰形成破坏，冰形成是由使用冷冻的更传统的冷冻保存方案引起的。玻璃体冷冻保存是一种替代的冷冻保存策略，其将组织稳定为玻璃，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。通过模仿某些昆虫在零度以下生存所采用的策略，可以通过降低冷冻保护剂浓度同时减少或抑制冰的形成来避免使用高浓度冷冻保护剂制剂。这些昆虫结合联合收割机增加冷冻保护剂含量（甘油）与寒冷环境条件的开始，产生几种抗冻肽（AFP）以在低至-80 °C的温度下生存。该可行性研究是为了确定使用这些昆虫AFP减少或抑制用于在常规条件下不能很好地冷冻保存的细胞的冷冻保存的冰形成的潜在益处。将研究这些AFP控制细胞内外冰的能力。然后将优化建立在冷冻保存后产生> 75%活力的方案并应用于II期组织。 公共卫生相关性：长期储存技术对于新开发的细胞疗法是必要的。冷冻保存是最明显的储存途径，但细胞和组织中的冰形成可能导致无法弥补的损害。使用高浓度的冷冻保护剂将有助于避免结冰，但以细胞毒性为代价。该提案将开发使用抗冻蛋白的保存方案，以便需要更少的冷冻保护剂，并最大限度地减少或消除冰的形成。这一策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。

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.