A NOVEL FROZEN STORAGE SYSTEM FOR CORD BLOOD STEM CELLS

描述（由申请人提供）：随着针对基础研究或临床应用的造血祖细胞（HPCs）纯化和/或体外扩增的努力不断增加，对这些细胞的改进、有效保存方法的开发需求也随之增加，以提供“细胞库”。此外，随着纯化祖细胞同种异体移植的增加，由于使用受污染的供体材料有传播疾病的可能性，因此只能使用经过传染病筛查的供体的细胞。储存冷冻细胞的能力将允许有足够的时间进行适当的筛选。因此，这项持续研究的总体目标是进一步开发和优化人类胎盘/脐带血（PCB）来源的造血祖细胞（HPCs）的冷冻保存和储存系统，特别是因为它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（移植）。在第一阶段，开发了一套系统，该系统提供了(1)一种高效、简化的大批量冷冻HPC装置方法，以及(2)在保持封闭系统的同时优化了冷冻保护剂（DMSO）的去除。在II期，该系统将被改进，以允许(1)在临床环境中易于使用，(2)易于制造/商业化。第二阶段提案的目标是基于健全的生物物理细胞类型特征和数学建模来完善封闭冷冻保存、储存和PCB洗涤系统，这将允许高效、安全地使用冷冻保存的PCB衍生HPCs。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-使用半固体培养基中的集落形成试验来确定克隆能力；(2)使用人类长期培养起始细胞（LTC-IC）试验来验证和改进；(3)通过冷冻/解冻/洗涤HPCs并移植到NOD/SCID小鼠模型中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.