THE EX VIVO EXPANSION OF CRYOPRESERVED CORD BLOOD

这项建议的目的是描述使用药剂级无血清培养液的培养条件，以支持用于成人移植的冷冻保存的脐带血的体外扩增。本文介绍的研究将使用最近开发的用于人类造血的宫内移植模型来评估保留短期和长期造血重建细胞所需的临床相关培养条件。本建议中提供的可行性数据表明，经冷冻保存的脐带血可以在专门为脐带血CD34+细胞体外扩增而设计的药剂级无血清培养液中进行体外扩增，2)保持其在绵羊模型中的长期移植能力，用于人类造血。在第一阶段的工作中，我们建议扩展这些研究，以评估各种细胞因子鸡尾酒、浓度效应和培养时间对冷冻保存的脐带血细胞体外扩增的作用。特别是，我们建议分析体外培养细胞的细胞周期状态，2)活细胞数，3)免疫表型，4)集落形成，5)长期起始细胞，6)长期移植能力。第二阶段将重点放在体外和体内造血试验中的其他细胞因子组合、浓度和培养时间，用于在人类造血的绵羊模型中长期植入和量化移植细胞的扩增潜力。使用上述方法，我们将能够以临床相关的方式解决一些关于成人移植中使用低温保存的脐带血的体外扩增的重要问题。拟议的商业应用：在成人移植中使用药剂级无血清试剂和条件对冷冻保存的脐带血进行体外扩增将是必不可少的。

The goal of this proposal is to delineate the culture conditions using pharmaceutical grade serum-free medium that will support the ex vivo expansion of cryopreserved umbilical cord blood for use in adult transplantation. The studies presented here will evaluate clinically relevant culture conditions necessary to retain the short-term and long-term hematopoietic reconstituting cells using the recently developed sheep in utero transplantation model for human hematopoiesis. Feasibility data presented in this proposal shows that l) cryopreserved cord blood can be ex vivo expanded in a pharmaceutical grade serum-free medium specifically designed for the ex vivo expansion of CD34+ cells from cord blood, and 2) maintain their long-term engrafting ability in the sheep model for human hematopoiesis. In the Phase I effort, we propose to extend these studies to evaluate the role various cytokine cocktails, concentration effects, and culture periods have on ex vivo expansion of cryopreserved cord blood cells. In particular, we propose to analyze the cultured cells in in vitro hematopoietic assays for I) cell cycle status, 2) viable cell number, 3) immunophenotype, 4) colony formation, 5) long- term initiating cells, and 6) long-term engrafting ability using the sheep model for human hematopoiesis. The Phase II will focus on other cytokine combinations, concentrations and culture times in the in vitro hematopoietic assays and in vivo for long-term engraftment and quantitation of the expansion potential of the engrafting cells in the sheep model for human hematopoiesis. Using the above approach we will be able to address, in a clinically relevant manner, a number of important issues concerning the use of ex vivo expansion of cryopreserved umbilical cord blood for adult transplantation. PROPOSED COMMERCIAL APPLICATION: The ex vivo expansion of cryopreserved cord blood with pharmaceutical grade serum-free reagents and conditions will be essential in its use in adult transplantation.