THE EX VIVO EXPANSION OF CRYOPRESERVED CORD BLOOD

本提案的目的是描述使用药用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离体扩增。本研究将使用最近开发的绵羊子宫内移植人造血模型，评价保留短期和长期造血重建细胞所需的临床相关培养条件。本提案中提供的可行性数据表明：1）冻存脐带血可在专门设计用于脐带血中CD 34+细胞体外扩增的药用级无血清培养基中进行体外扩增; 2）在绵羊模型中维持其长期移植能力，用于人类造血。在I期研究中，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冻存脐带血细胞体外扩增的作用。特别地，我们提出在体外造血测定中分析培养的细胞的I）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，和6）使用用于人造血的绵羊模型的长期移植能力。II期将重点关注体外造血试验中的其他细胞因子组合、浓度和培养时间，以及绵羊人造血模型中长期植入和植入细胞扩增潜力定量的体内试验。使用上述方法，我们将能够解决，在临床相关的方式，一些重要的问题，关于使用体外扩增的冷冻保存的脐带血成人移植。拟定商业应用：体外扩增冷冻保存的脐带血与药用级无血清试剂和条件将是必不可少的，在其使用在成人移植。

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.