SERUM-FREE CULTURE CONDITIONS FOR CELLS CAPABLE OF PRODUCING LONG-TERM SURVIVAL IN LETHALLY IRRADIATED MICE

The goal of ex vivo culture is to expand and/or differentiate cells in culture such that they retain their functional characteristics when r einfused into a patient. The studies presented here analyzed the use o f culture conditions devoid of serum to expand murine hematopoietic st em 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 c ultured for up to 21 days were assessed for granulocyte-macrophage col ony-forming cells (GM-CFC), spleen colony-forming units, and cells res ponsible for short-term and long-term hematopoietic repopulation in le thally irradiated mice. Compared to initial seeding levels, the presen ce of SCF and GM-CSF increased total cell numbers 90-fold and GM-CFC n umbers 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 rec onstitution were optimum with cells cultured for only seven days: surv ival 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. Hybri dization analysis of bone marrow, spleen and thymus DNA from irradiate d mice transplanted with these cultured cells confirmed male donor cel l-derived repopulation at 45 days and 180 days post-transplant. These studies illustrate that murine GM-CFM can be expanded and that long-te rm 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 t hat control hematopoietic stem cell proliferation.