title: Serum-free culture conditions for cells capable of producing long-term survival in lethally irradiated mice.
abstract: The goal of ex vivo culture is to expand and/or differentiate cells in culture such that they retain their functional characteristics when reinfused into a patient. The studies presented here analyzed the use of culture conditions devoid of serum to expand murine hematopoietic stem cells. Bone marrow cells from male B6D2F1/J mice were cultured for up to 28 days in serum-free medium in the absence or presence of stem cell factor (SCF), GM-CSF or a combination of the two factors. Cells cultured for up to 21 days were assessed for granulocyte-macrophage colony-forming cells (GM-CFC), spleen colony-forming units, and cells responsible for short-term and long-term hematopoietic repopulation in lethally irradiated mice. Compared to initial seeding levels, the presence of SCF and GM-CSF increased total cell numbers 90-fold and GM-CFC numbers 42-fold over a 21-28 day culture period. Although spleen colony-forming unit cells did not increase, they were maintained at initial seeding levels over a 21-day period in the presence of SCF and GM-CSF. In lethally irradiated mice, survival enhancement and hematologic reconstitution were optimum with cells cultured for only seven days: survival at six months was 100% with cells cultured in SCF plus GM-CSF or SCF alone, compared to 50% with cells cultured with only GM-CSF. Hybridization analysis of bone marrow, spleen and thymus DNA from irradiated mice transplanted with these cultured cells confirmed male donor cell-derived repopulation at 45 days and 180 days post-transplant. These studies illustrate that murine GM-CFC can be expanded and that long-term repopulating hematopoietic cells can, at the minimum, be maintained ex vivo in serum-free culture. The use of defined serum-free culture systems holds great promise for further evaluation of the mechanisms that control hematopoietic stem cell proliferation.
authors.affiliation: Quality Biological, Inc., Gaithersburg, Maryland 20879, USA.
authors.forename: R L
authors.initials: RL
authors.lastname: Brown
authors.forename: F S
authors.initials: FS
authors.lastname: Xu
authors.forename: S K
authors.initials: SK
authors.lastname: Dusing
authors.forename: Q
authors.initials: Q
authors.lastname: Li
authors.forename: R
authors.initials: R
authors.lastname: Fischer
authors.forename: M
authors.initials: M
authors.lastname: Patchen
mesh_terms: D000818:Animals; D001853:Bone Marrow / Q000528:radiation effects; D001854:Bone Marrow Cells; D018929:Cell Culture Techniques / Q000379:methods*; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D016207:Cytokines / Q000494:pharmacology; D004247:DNA / Q000032:analysis; D005260:Female; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D011833:Radiation Injuries, Experimental / Q000401:mortality / Q000628:therapy; D016019:Survival Analysis; D013997:Time Factors; D014916:Whole-Body Irradiation; D014998:Y Chromosome
chemical_list: D016895:Culture Media, Serum-Free; D016207:Cytokines; D004247:DNA