微囊微环境体外扩增人脐血造血干/祖细胞

In vitro expansion of umbilical cord blood hematopoietic stem/progenitor cells in microcapsule microenvironment

背景:由于单份脐血所含的造血细胞数量有限,目前只能用于儿童或低体质量成人血液病和急性辐射损伤等疾病患者,有效扩增脐血造血干/祖细胞已成为研究热点。目的:观察微囊微环境对脐血造血干/祖细胞扩增的影响,以及此过程中可否使造血干/祖细胞在扩增的同时仍维持其未分化状态。设计、时间及地点:单一样本观察,于2006-06/2007-09在中国科学院大连化学物理研究所完成。材料:正常足月产新生儿脐带血由大连市妇产医院提供,提供者知情同意。方法:Ficoll法梯度分离人脐血单个核细胞,采用静电液滴法在生理条件下进行微囊化包封和体外培养。以同条件平面培养的脐血单个核细胞作为对照。主要观察指标:观察微囊化脐血细胞的生长特点及脐血细胞总数的变化;流式细胞仪检测培养过程中CD34+细胞扩增情况;应用甲基纤维素半固体培养法观察扩增细胞的集落形成能力。结果:脐血细胞在微胶囊内持续增殖,并以聚集成团的三维方式生长,两种培养方式对脐血细胞总数均无明显影响(P>0.05)。对比CD34+细胞扩增和细胞集落的生成发现,微囊化培养脐血细胞的CD34+细胞数量和集落密度均在培养第6天达到高峰,明显高于平面培养3d时所达到的峰值;之后逐渐下降,至12d后平面培养细胞的CD34+细胞和集落形成能力几乎检测不到,而此时微囊化培养的CD34+细胞数量和集落密度仍与平面培养的扩增高峰相近。结论:微囊化培养能有效扩增人脐血造血干/祖细胞,并显著减缓造血干/祖细胞的分化进程,维持其多分化潜能,提示微囊为造血干/祖细胞维持未分化状态的扩增提供了特殊的微环境。

BACKGROUND： Cord blood contains limited population of hematopoietic cell, which only applied in treating hematonosis and acute radiation injury in children or adult with low body mass. Therefore, it is emergent to explore an effective method to expand human umbilical cord blood （UCB） hematopoietic stem/progenitor cells （HSCs）. OBJECTIVE： To evaluate the role of microcapsule microenvironment in the expansion of human UCB HSCs. DESIGN, TIME AND SETTING： A single sample observation was pedormed at the Dalian Institute of Chemical Physics, Chinese Academy of Sciences between June 2006 and September 2007. MATERIALS： The UCB was obtained from Dalian Gynecologic and Obstetric Hospital. All subjects gave written inform consent. METHODS： Human UCB mononuclear cells were purificated by the method of Ficoll density gradient separation, followed by encapsulating and culture in vitro with electrostatic drop generation technique. UCB mononuclear cells with Petri dish culture were served as control. MAIN OUTCOME MEASURES： The growth characteristics UCB cells in microcapsules and changes of total cellular score; the CD34＋ selected cells and the proliferation potential （the colony density） were examined by flow cytometry and methylcellulose semi solid culture method. RESULTS： UCB cells proliferated continually with three-dimensional growth in microcapsules. There was no difference in total cellular score in 2 groups （P 〉 0.05）. The expansion of CD34＋ selective cells and the colony density were reach a peak at day 6 in microcapsule system, which was significant greater than the peak value of cells in Petri dish reached at day 3, and then gradually decreased. At day 12, the CD34~ selective cells and the proliferation potential were could not detected in Petri dish, however, their level in microcapsules were similar to the maximums of cells in Petri dish. CONCLUSION： HSCs in microcapsule system can expand evidently and maintain their multi-potential continuously in vitro, which indicate that the microcapsule offer a microenvironment in maintaining HSCs expansion without differentiation.