To establish a practical and convenient method to expand hematopoietic cells (HCs), we applied chemically-fixed stromal cell layers formed within three-dimensional (3D) scaffolds to feeder of HC cultures. The HCs were expanded using two successive cultures. First, stromal cells were cultured within porous polymer scaffolds and formed tissue-engineered constructs (TECs); the scaffolds containing stromal cells, were fixed using aldehyde (formaldehyde or glutaraldehyde) or organic solvents (acetone, methanol or ethanol). Second, mouse fetal liver cells (FLCs), as a source of HCs, were cultured on the TECs for 2 weeks, and the effects of fixative solutions on expansion of primitive HCs (c-kit⁺ and CD34⁺ cells) were examined. In the cultures on aldehyde-fixed TECs, primitive HCs were expanded 2.5- to 5.1-fold in the cultures on TECs fixed with glutaraldehyde, whereas no expansions were detected in those fixed with formaldehyde. However, we achieved expansion of primitive HCs > fivefold in the cultures using TECs fixed with organic solvents. Among these solvents, the highest expansions—of roughly tenfold—were obtained using acetone fixation. Ethanol-fixed TECs also supported the expansion of the primitive HCs well (6.6- to 8.0-fold). In addition to these sufficient expansions, the procedure and storage of fixed TECs is fairly easy. Thus, HC expansion on chemically-fixed TECs may be a practical method for expanding primitive HCs.

为了建立一种实用且方便的方法来扩增造血细胞 (HC)，我们将在三维 (3D) 支架内形成的化学固定的基质细胞层应用于 HC 培养物的饲养层。HCs 使用两个连续的培养物进行扩增。首先，基质细胞在多孔聚合物支架内培养并形成组织工程结构（TECs）；含有基质细胞的支架用醛（甲醛或戊二醛）或有机溶剂（丙酮、甲醇或乙醇）固定。其次，小鼠胎肝细胞（FLCs）作为 HCs 的来源，在 TECs 上培养 2 周，固定液对原始 HCs（c-kit ⁺和 CD34 ^+）扩增的影响细胞）进行了检查。在醛固定的 TECs 上的培养物中，原始 HCs 在用戊二醛固定的 TECs 上的培养物中扩增了 2.5 到 5.1 倍，而在用甲醛固定的那些培养物中未检测到扩增。然而，我们在使用有机溶剂固定的 TECs 的培养物中实现了原始 HCs 的扩增 > 5 倍。在这些溶剂中，最高的膨胀率——大约十倍——是使用丙酮固定获得的。乙醇固定的 TEC 也很好地支持原始 HC 的扩展（6.6 到 8.0 倍）。除了这些足够的扩展之外，固定 TEC 的程序和存储也相当容易。因此，化学固定的 TECs 上的 HC 扩展可能是扩展原始 HCs 的实用方法。