The direct ex vivo expansion of hematopoietic stem cells (HSCs) extracted from umbilical cord blood (UCB) was investigated by a membrane filtration method using polyurethane foaming (PU) membranes with various nano-segments and pore sizes. After filtering UCB through the membranes and washing the membranes, the membranes were placed into culture medium, where the HSCs were expanded in a three-dimensional environment. The greatest expansion of HSCs was obtained through direct ex vivo expansion using PU membranes with nano-segments containing carboxylic acid. The membranes with larger pores (11 mu m) exhibited greater expansion of HSCs than the membranes with smaller pores (6 mu m), the membranes with nano-segments or unmodified membranes. The optimal pore size of the membranes was found to be regulated by the balance between the surface area required to trap HSCs and the diffusion potential of nutrition and growth factors, with a pore size of 11-100 mu m proving most effective in the direct ex vivo expansion method. HSCs expanded by the direct ex vivo expansion method retained the same pluripotency as HSCs purified by the conventional Ficoll-Paque method, followed by the MACS method, as measured by the colony-forming assay. The optimal washing solution for the membranes was also investigated, using diluted platelet-poor plasma with phosphate buffered solution for the direct ex vivo expansion of HSCs.
