Effect of pore diameter of a porous membrane on progenitor cell content during membrane-separated coculture of hematopoietic cells and a stromal cell line.

Abstract

The effect of pore diameter of a porous membrane on the progenitor cell content during a membrane-separated coculture of murine bone marrow hematopoietic cells and a murine stromal cell line, in which stromal cells adhered onto the lower surface of the membrane and hematopoietic cells were incubated on the upper surface of the membrane, was investigated in order to design a membrane bioreactor for ex vivo expansion of hematopoietic primitive cells employing exogeneic stromal cells. The hematopoietic progenitor cell [colony forming unit (CFU) Mix] content at 1 week in the membrane-separated coculture increased as the pore diameter of the membrane decreased from 12.0 to 0.4 microm. However, a further decrease in pore diameter from 0.4 to 0.1 microm did not affect the CFU-Mix content. Observation of stromal cells that adhered on the lower surface of the porous membranes (0.4 and 3.0 microm pore diameters) under a confocal scanning microscope after staining with rhodamine phalloidin suggested that stromal cells can migrate to the membrane's upper surface through pores with a diameter greater than 3.0 microm. Consequently, membrane having small (< or = 0.4 microm) pores that stromal cells could not pass through prevented direct contact between stromal and hematopoietic cells, which resulted in a higher content of hematopoietic progenitor cells (CFU-Mix) during the membrane-separated coculture.