Cytokine-augmented culture of haematopoietic progenitor cells in a novel three-dimensional cell growth matrix

Studies aimed at the in vitro expansion of haematopoietic progenitor cells (HPCs) have suffered from the conflict of increasing cell numbers while mai ntaining long-term repopulating ability. We have developed a long-term hone marrow bioreactor culture system resembling the marrow-microenvironment th at cultures HPCs in an inert, three-dimensional, porous biomatrix termed Ce llfoam (TM). Previous studies have shown that the short-term culture of CD3 4(+) cells in Cellfoam (TM) improved the maintenance and multipotency of ha ematopoietic stem cells compared to cells cultured on plastic dishes, In th is study, we examined the effects of low concentrations of cytokines includ ing stem cell factor (SCF), IL-3, and Flk-2/Flt-3 ligand, on the maintenanc e, preservation and multipotency of CD34(+) cells cultured for 3 or 6 weeks in Cellfoamo (TM). Analysis of cell yields using flow cytometry showed tha t in SCF and Flk-2/Flt-3 ligand-supplemented cultures as well as cytokine-f ree cultures, a higher number of CD45(+)34(+) and CD45(+)34(+)38(-)cells is observed in Cellfoam (TM) cultures as compared to plastic cultures. The fu nction of cultured cells was evaluated in colony-forming assays, The data d emonstrate that Cellfoam (TM) cultures supplemented with SCF and Flk-2/Flt- 3 ligand resulted in a higher output of colony activity compared to plastic cultures, Analysis of CAFC (29 days) activity also demonstrated that primi tive progenitors were maintained to a greater extent in Cellfoam (TM) cultu res containing either no cytokines or lo,v concentrations of early-acting c ytokines, These data suggest that culture of HPCs in three-dimensional bior eactors such as Cellfoam (TM) for extended periods may benefit from the add ition of low levels of early-acting cytokines, including SCF and Flk-2/Flt- 3 ligand, resulting in high yields of cells that are enriched for multipote nt haematopoietic progenitors, These findings demonstrate that a three-dime nsional matrix promotes the survival of primitive HPCs in culture and may m odulate the in vitro effects of cytokines. (C) 2001 Academic Press.