Expansion of mouse primitive hematopoietic cells in three-dimensional cultures on chemically fixed stromal cell layers.

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.