Miyoshi Hirotoshi, Morita Misa, Ohshima Norio, Sato Chiaki
Department of Biomedical Engineering, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan.
Department of Biomedical Engineering, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan.
Exp Hematol. 2015 Feb;43(2):115-24. doi: 10.1016/j.exphem.2014.08.008. Epub 2014 Nov 11.
To establish a highly efficient method of ex vivo expansion of hematopoietic cells (HCs), three-dimensional (3D) cocultures of HCs and stromal cell lines were performed using porous polymer scaffolds. Hematopoietic cells derived from mouse fetal livers were expanded by two successive cultures without the use of exogenous cytokines, namely, 3D cultures of stromal cells (DAS 104-8 cell line) to form stromal layers within the scaffolds, and, subsequently, by cocultures of the HCs on the stromal cell layers for 2 weeks. To expand the HCs more conveniently, in some experiments the stromal layers formed within the scaffolds were frozen (3D freezing) before the cocultures, then stored and applied to the cocultures after thawing. When the HCs were cocultured on the stromal layers of the DAS 104-8 cells, primitive HCs (c-kit(+) and CD34(+) cells) were expanded several fold during the cocultures. In contrast, the expansion of these primitive HCs was remarkably enhanced in the cocultures using the 3D frozen-thawed DAS 104-8 stromal layers (c-kit(+) cells > fifteenfold and CD34(+) cells > thirtyfold), and these expansions were significantly higher than those without the 3D freezing. The expansions enhanced by cocultures on the 3D frozen-thawed stromal layers were also observed in the cocultures with another stromal cell line (DAS 104-4). Because 3D frozen-thawed stromal cell lines are easy to handle, 3D coculture of HCs on frozen-thawed stromal cell lines may be an effective and convenient method for expanding primitive HCs.
为建立一种高效的造血细胞（HCs）体外扩增方法，使用多孔聚合物支架对HCs和基质细胞系进行了三维（3D）共培养。从小鼠胎肝中获取的造血细胞在不使用外源性细胞因子的情况下进行了两轮连续培养，即首先进行基质细胞（DAS 104 - 8细胞系）的3D培养，使其在支架内形成基质层，随后将HCs与基质细胞层共培养2周。为了更方便地扩增HCs，在一些实验中，将支架内形成的基质层在共培养前进行冷冻（3D冷冻），然后储存，解冻后应用于共培养。当HCs与DAS 104 - 8细胞的基质层共培养时，原始HCs（c-kit(+)和CD34(+)细胞）在共培养过程中扩增了数倍。相比之下，在使用3D冻融DAS 104 - 8基质层的共培养中，这些原始HCs的扩增显著增强（c-kit(+)细胞>15倍，CD34(+)细胞>30倍），且这些扩增明显高于未进行3D冷冻的情况。在与另一种基质细胞系（DAS 104 - 4）的共培养中也观察到，在3D冻融基质层上共培养可增强扩增效果。由于3D冻融基质细胞系易于操作，在冻融基质细胞系上对HCs进行3D共培养可能是一种有效且方便的扩增原始HCs的方法。
