Long-term cytokine-free expansion of cord blood mononuclear cells in three-dimensional scaffolds

Abstract

Cord blood expansion ex vivo can be achieved in liquid suspension through the addition of cytokines at the expense of often undesirable cell differentiation. In order to derive a cytokine-free dynamic culture system, we hypothesised that a three-dimensional (3D) environment in the form of highly porous scaffolds made of poly (D, L-lactide-co-glycolide) (PLGA) or polyurethane (PU) for the biomimetic growth of cord blood mononuclear cells (CBMNCs), would facilitate expansion of hematopoietic cells without exogenous cytokines. Both scaffolds supported cellular expansion ex vivo. Cytokine-free, long-term culture was best in PU coated with collagen type I (54-fold expansion). In contrast, traditional 2D well- plate cultures collapsed within 4 days in the absence of cytokines. CBMNCs cultured in the scaffolds were visualised by scanning electron microscopy and immunophenotypic/immunostaining analysis and the studies validated the presence of a dynamic culture containing erythroid precursors (CD45 /CD71þ/ CD235aþ), hematopoietic stem/progenitor cells (CD38 CD34þ, CD117þ), maturing myeloid cells (CD38þ, MPOþ), CD4þ and CD8þ T-lymphocytes and megakaryocytes (FVIIIþ). Colony forming unit (CFU) assays indicated that BFU-E and CFU-GM increased (p < 0.05) whereas CFU-GEMM were maintained at week 4. In conclusion, this 3D culture system is capable of long-term, cytokine-free expansion of CBMNCs, enabling the study of hematopoiesis and providing a potential platform for drug discovery and thera- peutic applications ex vivo.