3D culture of murine hematopoietic cells with spatial development of stromal cells in nonwoven fabrics.

BACKGROUND The in vivo hematopoietic microenvironment is composed of stromal cells and extracellular matrix in a 3D configuration. We have created a 3D microenvironment in vitro, employing spatial development of stromal cells in a nonwoven fabric porous carrier, Fibra-cel (FC). We compared its performance with that of a 2D microenvironment. METHODS Primary murine BM cells were inoculated on the layers of stromal cells prepared in FC (3D) or on a dish (2D) and cultured for 7-21 days. The hematopoietic cells harvested from the cultures were evaluated by colony-forming unit (CFU) assay and transplantation to sub lethally irradiated mice. RESULTS The maximum stromal cell concentration in the 2D culture was higher than that in the 3D culture. However, the hematopoietic cell concentration in the 3D culture was kept at a higher level than that in the 2D culture. The number of CFU-mix increased five times during 3D cultivation, but decreased in the 2D culture. The engraftment percentage of 3D cultured cells was comparable with that of fresh cells, and markedly higher than that of 2D cultured cells. DISCUSSION The 3D culture constructed with FC and stromal cells was clearly superior to 2D culture because hematopoietic progenitor cells were expanded without the addition of cytokines and the content of hematopoietic stem cells was maintained.