Development of the device for ex vivo expansion of cord blood stem cells and its clinical application

近年来，脐带血作为继骨髓和外周血之后的第三种造血干细胞来源受到关注。然而，脐带血最大的问题是它的局限性：所含造血干细胞的数量，这限制了脐带血对成人的管理。我们开发了一种独特的培养系统，其中脐带血和基质细胞被膜分离，但仍然通过0.45微米的孔保持接触。在实验中，我们能够用这个系统扩增脐带血干细胞。为了使其临床应用，我们必须做到1）控制来自小鼠基质细胞系的异种蛋白和微生物的污染，2）对培养系统进行安全化，3）评估植入和长期造血维持。出于这些目的，我们订购了生物风险公司Bioreliance Co.，参照美国食品药品监督管理局（FDA）的指导原则，对异种蛋白质和微生物的污染进行检查，证实小鼠间质细胞系HESS-5不含异种微生物。我们还开发了使用100 cm 2膜的大规模培养系统。我们用SRC定量分析法对SRC的扩增进行了评估，证实SRC在培养的5天内扩增了10倍以上。我们在IRB发布后开始了临床方案。

Recently, cord blood has been calling attention as a third source for supplying hematopoietic stem cells, next to bone marrow and peripheral blood. However, the biggest problem with cord blood is its limitation : the amount of hematopoietic stem cells contained, which restricts the administration of cord blood to adults. We developed a unique system for culture in which cord blood and stromal cells are separated by membrane, but still keep contact through pores with 0.45 micrometer. We were able to expand cord blood stem cells with this system in experiments. For the clinical application, we have to attain 1) control of the contamination of xenogeneic proteins and micro-organisms derived from murine stromal cell line, 2) sacale up the culture system, and 3) evaluation of the engraftment and long-term sustain of hematopoiesis. For these purposes we ordered the bioventure company, Bioreliance Co., to check the contamination of xenogeneic proteins and micro-organism in the reference of the guideline from FDA and confirmed the murine stromal cell line, HESS-5, was free from xenogeneic micro-organism. We also developed the large scale culture system by using membrane of 100 cm2. We evaluated the expansion of SRC by using of quantitative SRC assay, and confirmed more than 10 times expansion of SRC within 5 days of culture. We started clinical protocol after the disussion in IRB.