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Development of tissue-engineered artificial immune system

组织工程人工免疫系统的开发

基本信息

批准号：
15200033
负责人：
MIYOSHI Hirotoshi
金额：
$ 32.2万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15200033/
关键词：
hematopoietic stem cell stromal cell fetal liver cell artificial bone marrow growth differentiation three-dimensional culture porous scaffold tissue engineering 胎子肝臓細胞共培養シグナル分子

项目摘要

To develop a tissue-engineered artificial immune system, three-dimensional (3-D) coculture of hematopoietic cells and stromal cells were performed using porous polyvinyl formal (PVF) resin as a substrate material. In this series of culture experiments, mouse fetal liver cells (FLCs) were used as a hematopoietic cell source, and effects of stromal cell lines and 3-D cryopreservation of these cell lines on differentiation and expansion of hematopoietic stem cells were investigated.When FLCs were cocultured with DAS 104-8 stromal cell line, 3-D culture for 2 weeks resulted in 5-fold expansion in hematopoietic progenitor cells (HPCs). To expand HPCs more conveniently, 3-D cryopreserved stromal cells which were made by cryopreserving stromal cells immobilized within the PVF resin cubes, were applied to the cultures of hematopoietic cells. After 2 weeks of coculture experiments, HPCs were expanded over a 15-fold, and these efficiencies were far better than the cultures using stromal cells without cryopreservation. Similar results were obtained when another stromal cell line (DAS 104-4) was used. Moreover, in the culture experiments using DAS 104-8 of which growth was suppressed by mitomycin C treatment, higher (15-fold) expansion of HPCs was also achieved than those using untreated DAS 104-8. Therefore, it was clarified that efficiencies of HPC expansion were improved by applying 3-D cryopreserved stromal cell lines mainly due to the suppressed growth of these cell lines.To facilitate specific differentiation of HPCs into B cell lineage, FLCs were cocultured with DAS 104-8 cell line in the presence of interleukin-7. However, numbers of B cells as well as HPCs in the PVF cubes decreased rapidly with elapse of culture period. Therefore, further investigations concerning differentiation into B cells will be required.In conclusion, efficient expansion system of HPCs was established by using 3-D cryopreserved stromal cells.

为了开发组织工程化人工免疫系统，使用多孔聚乙烯醇缩甲醛（PVF）树脂作为基质材料进行造血细胞和基质细胞的三维（3-D）共培养。本实验以小鼠胎肝细胞（FLC）为造血干细胞来源，研究了基质细胞系和细胞系三维冻存对FLC分化和扩增的影响，FLC与DAS 104-8基质细胞系共培养时，3-D培养2周导致造血祖细胞（HPC）扩增5倍。为了更方便地扩增造血干细胞，将冻存的基质细胞固定在PVF树脂立方体中，制成三维冻存的基质细胞，应用于造血细胞的培养。经过2周的共培养实验，HPCs扩增超过15倍，这些效率远远优于使用基质细胞未经冷冻保存的培养物。当使用另一种基质细胞系（DAS 104-4）时，获得了类似的结果。此外，在使用通过丝裂霉素C处理抑制生长的DAS 104-8的培养实验中，也实现了比使用未处理DAS 104-8的那些更高（15倍）的HPC扩增。为了促进HPC向B细胞系的特异性分化，将FLC与DAS 104-8细胞系在IL-7存在下共培养。然而，随着培养时间的推移，PVF立方体中的B细胞以及HPC的数量迅速下降。因此，在向B细胞分化方面还需要进一步的研究。总之，利用三维冻存的基质细胞，建立了高效的体外扩增体系。