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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.

Serum‐Free Culture Conditions for Cells Capable of Producing Long‐Term Survival in Lethally Irradiated Mice