A three-dimensional perfusion bioreactor system with porous glycosaminoglycan-based scaffolds for expansion of CD34+ hematopoietic stem cells

In vitro expansion of hematopoietic stem cells (HSCs) has been used as an approach to obtaining sufficient numbers of stem cells for successful engraftment of HSC for transplantation. Glycosaminoglycans (GAGs) are anionic polysaccharides of extracellular matrix that have been shown to influence the proliferation and differentiation of HSCs. Our culture studies demonstrated that specific GAGs and GAG analogues enhanced the proliferation of cord blood CD34+ cells, when covalently immobilized on chitosan membranes. In particular, it was possible to achieve high levels of proliferation by HSCs with minimal differentiatioe activity. However, the culture results were limited with respect to cell density and control of environmental parameters such as oxygen level and pH. A three-dimensional perfusion bioreactor system with GAG-chitosan scaffold was designed to more closely mimic the bone marrow microenvironment while providing more precise control of environmental parameters and achieving the high yield expansion of HSCs. CD34+ HSCs were isolated from umbilical cord blood by immunomagnetic separation and cultured within the porous scaffold in a perfusion bioreactor system. The scaffolds were harvested for SEM and histology analysis. Cellular phenotype and HSC characteristics were evaluated via flow cytometry and colony forming unit assays. The results indicate good cell retention and proliferation within the perfused scaffolds. Cell proliferation also correlates well with the parallel dish cultures. Oxygen consumption in the perfusion bioreactor system increased constantly during the culture period, indicating the increase of total number of cells. Perfusion cultures within scaffolds show higher surface antigen expressions of primitive progenitors and committed progenitor cells as compared to static monolayer culture. In colony assay, cells from perfusion culture exhibited much better performance than static one. Perfusion culture at low oxygen (5%) enhanced the expansion of CD34+ cells and colony-forming activity compared to high oxygen (19%). In this study, we demonstrate a three-dimensional perfusion culture within GAG-immobilized scaffolds provides improved maintenance and expansion of HSCs, especially in low oxygen similar to in vivo bone marrow microenvironment.