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

Abstract

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.

Fig. 1

SEM micrographs of the PUF-W1 scaffolds: a side cross sectional view, b top cross sectional view

Fig. 2

A schematic diagram of the 3D culture configuration in PUF pores

Fig. 3

Hematopoietic cell proliferation. a Produced Cell counts in each culture. Each 3 days old culture medium containing nonadherent cells was replaced, and cells were counted as Produced cells (n = 3 by 42 day, then, n = 1). b Cumulative cell production in each culture. c Adherent Cell counts at each 7 days, which adhered to PUF surfaces (n = 2). The symbols: (triangle) W1 culture, (circle) R4 culture, and (square) monolayer culture

Fig. 4

The expression of cell surface markers by mBMMNCs and Produced Cells in PUF culture at day 21–24. The cells were harvested from 18 wells and used for these evaluations. (open) W1 culture, (hatched) R4 culture, and (closed) fresh BMMNC

Fig. 5

At day 32, SEM micrographs of adherent cells from mBMMNC culture on (a) PUF-W1 and (b) PUF-R4. It was observed that the stromal cells have spread into the PUF scaffolds (arrows). On the other hand, the extension from like fibroblast cells was not observed

Fig. 6

Comparison in number of colony forming units in W1 culture, R4 culture and monolayer culture on day 14 and 21 (n = 4). The bars: (open) W1 culture, (hatched) R4 culture, and (closed) monolayer culture