IMPROVED CONDITIONS FOR HUMAN HEMATOPOIETIC CELL CULTURE

改善人类造血细胞培养条件

• 批准号: 2224348
• 负责人: Eleftherios T Papoutsakis
• 金额: $ 17.43万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2224348
• 关键词: acidity /alkalinity; biomaterial development /preparation; bioreactors; cell adhesion; cell cycle; cell differentiation; colony stimulating factor; cytokine; drug interactions; free radical oxygen; hematopoiesis; hematopoietic stem cells; human tissue; interleukin 1; interleukin 3; interleukin 6; northern blottings; oxygen tension; polymerase chain reaction; temperature; tissue /cell culture; transforming growth factors; tumor necrosis factor alpha; umbilical cord

Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, the painful bone marrow harvest procedure, autologous (patient) marrow that is depleted from prior therapy or has leukemic involvement, and the small number of progenitor cells in umbilical cord blood. An in vitro system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplantation, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow required for transplantation, and allow collection of peripheral blood stem and progenitor cells to replace the bone marrow harvest process. Present methods for long-term bone marrow (hematopoietic) culture (LTHC) on stromal layers in flasks lack a well-controlled growth environment. The proposed research will improve the prospects for use of LTHC in bone marrow transplantation by identifying conditions that allow for rapid expansion of cord blood hematopoietic progenitors in a perfusion bioreactor without the need for stromal cells. Eliminating the stromal layer will eliminate the need for a stomal cell donor, reduce culture time, and simplify culture conditions. Existing perfusion chambers suitable for culture of cells on a stromal layer will be modified for operation without stromal cells by adding a nylon mesh to better retain nonadherent cells. Analysis of the bone marrow environment suggests that further improvements in progenitor cell expansion may be obtained by manipulating the culture oxygen (O2) tension, pH, and temperature (T), and by developing more effective combinations of cytokines for LTHC. Progenitor cell expansion is enhanced at reduced O2 tensions. The effects of culture in medium saturated with 2.5-10% O2 will be examined via assays for colony-forming cells and the primitive long-term culture- initiating cells (LTC-IC). Mechanisms responsible for enhanced hematopoiesis at reduced O2 tension will be investigated by monitoring levels of oxidizing species and cytokines in the medium. The cytokine mixture IL-3/IL-6/stem cell factor, which has proved successful in expanding primitive cells, will be used as the base formulation to test the effectiveness of other cytokine combinations which will include both positive (IL-1,G-CSF) and negative (TNF-alpha, TGF-Beta) regulators of hematopoiesis. These cytokine combinations will be tested first in a static suspension culture assay. The best combinations will be then examined under perfusion conditions. While 33 degrees C is generally considered better than 37 degrees C for static LTHC, this may be due to less rapid nutrient depletion at 33 degrees C in these infrequently- fed cultures. We will compare perfusion cultures at 35 degrees C and 37 degrees C against the control 33 degrees C culture for effects on progenitor cell expansion. pH effects have not been examined in LTHC even though extracellular and/or intracellular pH are important regulators of proliferation, function and differentiation for a variety of cell types. Cultures at three pH values from 6.9 to 7.8 will be compared against the control culture at pH 7.35.

用于骨髓移植的造血细胞的来源是 受相容供体供应的限制，病毒感染的可能性 感染，痛苦的骨髓采集程序，自体 （患者）骨髓从既往治疗中耗尽或患有白血病 参与，和少量的祖细胞在脐带 血 造血祖细胞体外扩增系统的建立 可以增加有资格接受自体移植的患者数量， 移植，允许使用脐带血造血细胞， 重新繁殖成年人，减少骨髓的数量， 移植，并允许收集外周血干细胞， 祖细胞来代替骨髓收获过程。 本 长期骨髓（造血）培养（LTHC）的方法 烧瓶中的基质层缺乏良好控制的生长环境。 的 拟议的研究将改善LTHC在骨骼中的应用前景 骨髓移植通过识别允许快速 脐带血造血祖细胞的扩增 不需要基质细胞的生物反应器。 消除基质 层将消除对造口细胞供体的需要，减少培养 时间，简化培养条件。 现有灌注室 适合于在基质层上培养细胞的方法将被修改， 手术中不加基质细胞，通过尼龙网更好的保留 非贴壁细胞 骨髓环境分析表明， 祖细胞扩增的进一步改进可通过以下方法获得： 操纵培养物氧（O2）张力、pH和温度（T）， 并通过开发更有效的细胞因子组合来治疗LTHC。 祖细胞扩增增强在降低O2紧张。 的 将检查在2.5- 10%O2饱和的培养基中培养的效果 通过对集落形成细胞和原始长期培养物的分析， 启动细胞（LTC-IC）。 负责加强 将通过监测来研究氧分压降低时的造血 培养基中氧化物质和细胞因子的水平。 细胞因子 IL-3/IL-6/干细胞因子的混合物，已被证明在 扩大原始细胞，将被用作基础配方，以测试 其他细胞因子组合的有效性， 阳性（IL-1，G-CSF）和阴性（TNF-α，TGF-β）调节剂 造血 这些细胞因子组合将首先在一个 静态悬浮培养测定。 最好的组合是 在灌注条件下检查。 33摄氏度通常是 认为优于 静态LTHC为37 ℃，这可能是由于 在33摄氏度下，这些罕见的营养素消耗速度较慢- 饲养培养物。 我们将比较35 ℃和37 ℃下的灌注培养 33 ℃培养物的影响 祖细胞扩增。 在LTHC中尚未检查pH影响 尽管细胞外和/或细胞内pH是重要的 调节增殖，功能和分化的各种 细胞类型。 将在6.9至7.8的三个pH值下培养 与pH 7.35的对照培养物相比。