Development of tissue-engineered artificial immune system

为构建组织工程化人工免疫系统，以多孔聚乙烯醇缩甲醛(PVF)树脂为基质材料，进行了造血细胞与基质细胞的三维共培养。本实验以小鼠胎肝细胞为造血细胞来源，研究了基质细胞系及其三维冷冻保存对造血祖细胞分化和扩增的影响。为了更方便地扩增HPC，将PVF树脂块中固定的基质细胞进行三维冷冻保存，将其应用于造血细胞的培养中。经过2周的共培养实验，HPC扩增了15倍以上，这些效率远远好于使用未冷冻的基质细胞培养。用另一种基质细胞系(DAS 104-4)也得到了类似的结果。此外，在用丝裂霉素C抑制生长的DAS 104-8的培养实验中，HPC的扩增也比未处理的DAS 104-8高15倍。为了促进HPC向B细胞方向分化，将其与DAS 104-8细胞在白介素7的作用下进行共培养。然而，随着培养时间的延长，PVF中的B细胞和HPC数量迅速减少。综上所述，利用三维冷冻保存的基质细胞建立了高效的骨髓基质细胞扩增体系。

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.