THE EX VIVO EXPANSION OF CRYOPRESERVED CORD BLOOD

本提案的目标是描述使用药物级无血清培养基的培养条件，该培养基将支持冷冻保存的脐带血用于成人移植的体外扩增。本文提出的研究将评估临床相关的培养条件，以保留短期和长期造血重建细胞，使用最近开发的绵羊子宫移植模型用于人类造血。本研究提出的可行性数据表明，1)冷冻保存的脐带血可以在专门为脐带血CD34+细胞体外扩增设计的药物级无血清培养基中体外扩增，2)在人造血羊模型中保持其长期移植能力。在I期工作中，我们建议扩展这些研究，以评估各种细胞因子混合物，浓度效应和培养时间对低温保存脐带血细胞体外扩增的作用。特别是，我们建议在体外造血实验中分析培养的细胞：1)细胞周期状态，2)活细胞数量，3)免疫表型，4)集落形成，5)长期起始细胞，6)长期移植能力，使用羊模型进行人造血。II期将重点关注其他细胞因子的组合、浓度和体外造血试验的培养时间，以及体内长期移植和定量移植细胞在羊模型中用于人类造血的扩展潜力。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使用冷冻脐带血体外扩增用于成人移植的重要问题。建议的商业应用：用药物级无血清试剂和条件对冷冻保存的脐带血进行体外扩增在成人移植中使用是必不可少的。

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.