Ex vivo expansion of haematopoietic stem cells in a HFBR

Abstract

The transplantation of ex vivo expanded mobilized peripheral blood haematopoietic stem cells (PBSC), in place of unmanipulated cells following high dose chemotherapy, reduces the period of cytopenia associated with the therapy’s hemotoxicity. In this thesis the development and optimization of a preclinical prototype hollow fiber bioreactor (HFBR) for the ex vivo expansion of PBSC is described. Mass transport measurements and model of metabolite profiles demonstrate that Cuprophan and Polyflux are suitable membrane material for high-density cell expansion in a HFBR. Materials selected for the HFBR were found to be non-toxic following a 20-day saline extraction. Growth factor (GF) adsorption to the Polyflux membrane makes it unsuitable for expansion of GF dependent cells. However, the GF retention and minimal adsorption characteristics of the Cuprophan membrane are appropriate for this application.

Cell-free medium degrades at 37ºC by an oxygen dependent process generating byproducts that inhibit cell growth. This process is relevant to perfusion bioreactors where the bulk of the medium is maintained at 37ºC and is cell-free. Albumin was shown to slow the degradation process but was itself degraded by shear damage inflicted during recirculation. Treating recirculating medium with dialysis against albumin was shown to be a more effective way to mitigate the effects of degradation and lengthen the functional life of albumin over conventional suspension of albumin in the recirculating medium.

The preclinical prototype HFBR utilised dialysis against albumin to expand KG-1a cultures from densities as low as 3.5x10^5 cells/ml up to as high as 2x10^8 cells/ml with expansion rates equivalent to T-flask cultures. This process was then applied to PBSC where the targeted 100-fold expansion was achieved. Process optimization was continued using cord blood (CB) CD34+ cells. Growth factor loading sufficient for PBSC expansion in the HFBR was inadequate for CB expansions due to greater than anticipated CB uptake rates. The cell product from the HFBR contained significantly greater yields of CD34+ cells than attained using T-flask cultures. The HFBR platform is suitable for PBSC expansion and appears promising for CB expansion.