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Hematopoietic stem cells exist in specific niches in the bone marrow, and generate either more stem cells or differentiated hematopoietic progeny. In such microenvironments, cell-cell and cell-matrix interactions are as important as soluble factors such as cytokines. To provide a similar environment for in vitro studies, a three-dimensional culture technique is necessary. In this manuscript, we report the development of a three-dimensional culture system for murine bone marrow mononuclear cells (mBMMNCs) using polyurethane foam (PUF) as a scaffold. The mBMMNCs were inoculated into two kinds of PUF disks with different surface properties, and cultured without exogenous growth factors. After seeding the inside of the PUF pores with mBMMNCs, PUF disks were capable of supporting adherent cell growth and continuous cell production for up to 90 days. On days 21-24, most nonadherent cells were CD45 positive, and some of the cells were of the erythroid type. From comparisons of the cell growth in each PUF material, the mBMMNC culture in PUF-W1 produced more cells than the PUF-R4 culture. However, the mBMMNC culture in PUF-W1 had no advantages over PUF-R4 with regard to the maintenance of immature hematopoietic cells. The results of scanning electron microscopy and colony-forming assays confirmed the value of the different three dimensional cultures.

In vitro reconstruction of a three-dimensional mouse hematopoietic microenvironment in the pore of polyurethane foam