AbstractAbstract 828Human umbilical cord blood (CB) is a good source of hematopoietic stem cells (HSCs), but its use is limited by the low volume of the product and the relatively small number of HSCs. Conventional attempts at ex vivo expansion of HSCs and other hematopoietic cells have used two-dimensional (2D) tissue culture flasks or well-plates, which require feeder cells and/or various combinations of cytokines for long-term hematopoiesis. These conditions differ drastically from that provided by the natural bone marrow (BM) microenvironment wherein a complex network within the stem cell niche modulates self-renewal and pluripotency. Moreover 2D cultures have other limitations including lack of mixing, critical concentration gradients for pH, dissolved oxygen, cytokines, nutrients and metabolites, and the low number of cells that can be supported by a given surface area. Previously, we have shown that CB mononuclear cells (MNCs) can be expanded in a three dimensional (3D) static culture in cytokine-free medium for 4 weeks with a total expansion of 53-fold using polyurethane (PU) scaffolds coated with collagen type I. We hypothesized that combining these scaffolds with a perfused dual hollow fibre bioreactor (DHFB) would form a functional automatable BM biomimicry that could sustain long-term hematopoiesis and enhance the expansion of CBMNCs in a cytokine-free environment. The bioreactor includes a) a selective ceramic hollow fibre membrane system, which enables fast-flow perfusion with fresh medium for the supply of nutrients and removal of metabolites, b) a slow-flow, selectively-permeable membrane system for the supply of cytokines and the continuous harvest of mature red blood cells (RBCs) and, c) a PU scaffold rendered bioactive by collagen coating which provides a 3D in vitro culture system mimicking the natural BM microenvironment. CBMNCs were isolated using Ficoll-Paque density centrifiugation and seeded in the bioreactor at a density of 1.6×107cells/cm3. The DHFB was perfused at a flow rate of 160 ml/day in recirculation mode in order to allow for the accumulation of products. Nutrients and metabolites were monitored in the cell culture including glucose, glutamine, glutamate, lactate and ammonia and, the data used to perfuse the DHFB at optimal conditions. The hollow-fibre membranes allowed the selective mass transport of nutrients (glucose), metabolites (lactate), proteins (albumin) as well as RBCs. CBMNCs were cultured without the addition of exogenous cytokines for 3 weeks inside the DHFB, within which time cells were expanded by 300-fold, compared with 53-fold in 3D static culture correlates. Immunophenotypic analysis of extracted cells after 3 weeks showed an increase of CD71+CD45- erythroid progenitors as well as a maintained CD34+ hematopoietic progenitor cell population. Within the perfused DHFB, cells migrated into the pores of the scaffold establishing “niche-like” areas of growth identified by scanning electron microscopy, similar to those observed in static 3D culture conditions. After the 3D DHFB cultures had established, we “flexed” the system with the addition of 100ng/ml stem cell factor (SCF) and 2U/ml erythropoietin (EPO) in order to expand and mature erythrocytes and attempt selective harvesting via the closed-loop automated DHFB system. With this cocktail, mature enucleated RBCs were observed exiting the scaffold through the selectively permeable membrane, whilst other cell types and progenitors were maintained within the scaffold. This observation was confirmed by surface immunophenotype of the cells collected in the harvesting system. In conclusion, we have shown that the 3D DHFB system allowed for higher cytokine-free expansion of CBMNCs than that observed in the static 3D correlate, with evidence of directed differentiation of HSCs into erythroid progenitors in the absence of exogenous cytokines. This expansion and selective harvesting of RBCs was accomplished in a closed-loop system with monitoring and control optimisation of nutrient-waste exchange. The erythroid maturation and selective harvesting of RBCs was enhanced by the addition of SCF and EPO to the culture system. This technology enables a functional BM biomimicry and could be used as a tool to study the hematopoietic microenvironment in 3D ex vivo and for the eventual expansion of hematopoietic cell components for therapeutic use. Disclosures:No relevant conflicts of interest to declare.
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