Method and compositions for obtaining mature dendritic cells
View Patent ↗We describe an improved method for generating sizable numbers of mature dendritic cells from nonproliferating progenitors in human blood. The first step or “priming” phase is a culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4 to produce immature dendritic cells. The second step or “differentiation” phase requires the exposure to dendritic cell maturation factor such as monocyte conditioned medium. Using this two-step approach, substantial yields are obtained. The dendritic cells derive from this method have all the features of mature cells. They include a stellate cell shape, nonadherence to plastic, and very strong T cell stimulatory activity. The mature dendritic cells produced according to this invention are useful for activating T cells.
1. An in vitro method of producing stable mature CD83 positive dendritic cells from substantially CD83 negative immature dendritic cells which are derived from PBMC pluripotential cells having the potential of expressing either macrophage or dendritic cell characteristics, said method comprising the steps of:
(a) culturing a population of peripheral blood mononuclear cells comprising pluripotential cells which are CD14 positive and substantially CD83 negative with at least one cytokine to produce a population of immature dendritic cells characterized as substantially CD14 and CD83 negative; and
(b) culturing said substantially CD83 negative immature dendritic cells in the presence of a composition selected from the group consisting of fixed Staphylococcus aureus (SACS), peripheral blood mononuclear cell conditioned medium, and monocyte conditioned medium, in an amount and for a time sufficient for said immature dendritic cells to express characteristics of mature dendritic cells, wherein said characteristics of mature dendritic cells include an increase in CD83 and CD11c expression, a decrease in CD68 expression, and persistence of non-adherence for three days following removal of said composition from said culture of dendritic cells.
2. The method according to claim 1 , wherein the composition is monocyte conditioned medium.
3. The method according to claim 1 , wherein the cytokine is a combination of GM-CSF and either one or both of IL-4 and IL-13.
4. The method according to claim 3 , wherein said pluripotential cells are human.
5. The method according to claim 4 , wherein said pluripotential cells are obtained from human blood.
6. The method according to claim 5 , wherein the cytokine is a combination of GM-CSF and IL-4.
7. The method of claim 1 , wherein said composition is peripheral blood mononuclear cell conditioned medium.
8. The method of claim 1 , wherein said composition is macrophage conditioned medium.
9. The method of claim 1 , wherein said composition is fixed Staphylococcus aureus (SACS).
10. The method of claim 1 , wherein said immature dendritic cells are obtained by culturing a population of peripheral blood mononuclear cells with at least one cytokine in medium which does not contain fetal calf serum.