A NOVEL FROZEN STORAGE SYSTEM FOR CORD BLOOD STEM CELLS

描述(申请人提供)：随着基础研究或临床应用中对造血祖细胞(HPC)的纯化和/或体外扩增的努力日益增加，对这些细胞开发改进的、有效的保存方法以提供“细胞库”的需求也相应增加。此外，随着纯化的祖细胞同种异体移植的增加，使用受污染的捐赠者材料可能导致疾病传播，这要求只使用来自传染病筛查捐赠者的细胞。储存冷冻细胞的能力将为进行充分的筛查提供足够的时间。因此，这项持续研究的总体目标是进一步开发和优化一种改进的人胎盘/脐带血(PCB)来源的造血祖细胞(HPC)的冷冻保存和存储系统，特别是涉及到改善冻融细胞的正常结构和功能的保留，以改善临床结果(植入)。在第一阶段，开发了一种系统，该系统提供(1)一种高效、简化的散装冻结HPC装置的方法，以及(2)在保持封闭系统的同时优化移除低温保护剂(DMSO)。在第二阶段，这一系统将得到改进，以(1)易于在临床环境中使用，以及(2)易于制造/商业化。该第二阶段提案的目标是根据合理的生物物理细胞类型特定特性和数学模型来改进封闭式低温保存、存储和印刷电路板清洗系统，以允许高效和安全地使用低温保存的多氯联苯来源的高性能碳化物。为了实现这一目标，提出了以下具体目标：(1)验证和改进冷冻保存方法/系统-在半固体介质中使用集落形成实验来确定克隆形成能力，(2)使用人类长期培养启动细胞(LTC-IC)实验来验证和改进冷冻保存系统，以及(3)通过冷冻/解冻/清洗HPC并将其移植到NOD/SCID小鼠模型来测试冷冻保存系统。

DESCRIPTION (provided by applicant): With increasing effort directed toward purification and/or in vitro expansion of hematopoietic progenitor cells (HPCs) for either fundamental research or clinical applications, there is an associated, increased need for the development of improved, efficient preservation methods for these cells to provide "cell banks." In addition, with the increase in allograft of purified progenitor cells, the possibility of disease transmission from use of contaminated donor material requires that only cells from infectious disease-screened donors be used. The ability to store frozen cells would allow sufficient time for adequate screening to be performed. Therefore, the overall goal of this continuing research is to further develop and optimize an improved system for cryopreservation and storage of human placental/umbilical cord blood (PCB) derived hematopoietic progenitor cells (HPCs), especially as it relates to improving the retention of normal structure and function of frozen-thawed cells to improve clinical outcome (engraftment). In Phase I, a system was developed which provides (1) an efficient, simplified method of bulk-freezing HPC units, and (2) optimized removal of cryoprotectant (DMSO) while maintaining a closed system. In Phase II, this system will be refined to allow (1) ease of use in the clinical environment, and (2) ease of manufacture/commercialization. It is the goal of this Phase II proposal to refine the closed cryopreservation, storage, and PCB washing system based on sound biophysical cell-type specific characteristics and mathematical modeling which will allow efficient and safe use of cryopreserved PCB derived HPCs. To achieve this goal, the following specific aims are proposed: (1) Validation and refinement of the cryopreservation method/system-using colony forming assays in semi-solid media to determine clonogenic ability, (2) Validation and refinement using a human long-term culture initiating cell (LTC-IC) assay, and (3) Testing of the cryopreservation system by freezing/thawing/washing HPCs and transplanting into a NOD/SCID mouse model.