doi:10.3850/978-981-08-7615-9_TE06

ABSTRACT

The native bone marrow niche where hematopoietic cells reside consists of an intricate three-dimensional (3D) network of the extracellular matrix (ECM), hematopoietic cells, and soluble factors. However, in vitro studies performed traditionally on tissue culture plastics are two-dimensional (2D) in nature and misrepresents the study of cell-cell and cell-matrix interactions. In this work, we evaluated the use of highly porous Ca-alginate hydrogels in supporting the growth and differentiation of hematopoietic cells using the erythroleukemia cell line K562 (ATCC) as a cell model. Our results showed that at different initial seeding densities (1E5 – 5E5 cells/ml), expansion capacity was more consistent in 3D culture being independent of seeding densities and the population doubling time reduced by half in 3D compared to 2D culture. In optimizing the bead size and alginate concentration, the smallest bead ( < 1.8 μm) and lower alginate concentration (1.0%) yield the best cell growth. When additional ECM proteins were added into the Ca-alginate hydrogel, we observed greater responses in cell growth and differentiation in 3D culture but not in 2D culture. Wright-Giemsa stained cells in alginate beads containing collagen, fibronectin and laminin (5 μg/ml) at day 11 revealed that each protein has a specific effect on differentiation and maturation of K562 cells. Megakaryocytes were found in 3D culture containing laminin while neutrophilic band cells were found in presence of collagen. Cells in presence of fibronectin in 3D culture were mostly early myeloblasts. Taken together, we have demonstrated that the Ca-alginate hydrogel system is able to enhance growth and induce differentiation of erythroleukemic cells in the presence of specific proteins. The simple procedure makes this system an effective and promising in vitro model for studying hematopoietic stem cell behaviors with the potential for recapitulating the bone marrow niche.

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