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 maintaining long-term repopulating ability. We have developed a long-term bone marrow bioreactor culture system resembling the marrow-microenvironment that cultures HPCs in an inert, three-dimensional, porous biomatrix termed Cellfoam. Previous studies have shown that the short-term culture of CD34(+)cells in Cellfoam improved the maintenance and multipotency of haematopoietic stem cells compared to cells cultured on plastic dishes. In this study, we examined the effects of low concentrations of cytokines including stem cell factor (SCF), IL-3, and Flk-2/Flt-3 ligand, on the maintenance, preservation and multipotency of CD34(+) cells cultured for 3 or 6 weeks in Cellfoam. Analysis of cell yields using flow cytometry showed that in SCF and Flk-2/Flt-3 ligand-supplemented cultures as well as cytokine-free cultures, a higher number of CD45(+)34(+) and CD45(+)34(+)38(-) cells is observed in Cellfoam cultures as compared to plastic cultures. The function of cultured cells was evaluated in colony-forming assays. The data demonstrate that Cellfoam 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 primitive progenitors were maintained to a greater extent in Cellfoam cultures containing either no cytokines or low concentrations of early-acting cytokines. These data suggest that culture of HPCs in three-dimensional bioreactors such as Cellfoam for extended periods may benefit from the addition 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 multipotent haematopoietic progenitors. These findings demonstrate that a three-dimensional matrix promotes the survival of primitive HPCs in culture and may modulate the in vitro effects of cytokines.
Banu
N
N
Cytomatrix, Cell Growth Systems, Woburn, MA 01801, USA.
Rosenzweig
M
M
Kim
H
H
Bagley
J
J
Pykett
M
M
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000378:metabolism; D000943:Antigens, Differentiation / Q000378:metabolism; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002478:Cells, Cultured; D003470:Culture Media; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D007377:Interleukin-3 / Q000494:pharmacology; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008024:Ligands; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000494:pharmacology; D051379:Mice; D009244:NAD+ Nucleosidase / Q000378:metabolism; D019089:Stem Cell Factor / Q000494:pharmacology; D013997:Time Factors
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D003470:Culture Media; D007377:Interleukin-3; D008024:Ligands; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D017493:Leukocyte Common Antigens; D036541:ADP-ribosyl Cyclase; C498155:Cd38 protein, mouse; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1