Methods 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 for producing stable mature dendritic cells from pluripotential cells, comprising:
a) contacting said pluripotential cells having the potential of expressing either macrophage or dendritic cell characteristics with a combination of GM-CSF and IL-4 or IL-13 for a time sufficient to produce immature dendritic cells, and
b) adding a composition selected from the group consisting of peripheral blood mononuclear cell conditioned medium, monocyte conditioned medium and fixed Staphylococcus aureus Cowan 1 strain (SACS) to the immature dendritic cells produced in step (a) and culturing the cells for a time sufficient for the immature dendritic cells to produce stable mature dendritic cells that express a characteristic of mature dendritic cells,
wherein the characteristic is selected from the group consisting of increased CD83 expression, increased CD86 expression, decreased CD115 expression, and decreased CD32 expression relative to the immature dendritic cells.
2. The method of claim 1 , wherein the pluripotential cells are CD14 positive mononuclear pluripotential cells.
3. The method of claim 1 , wherein the pluripotential cells are peripheral blood mononuclear cells.
4. The method of claim 1 , wherein the pluripotential cells are monocytes.
5. The method of claim 1 , wherein the composition further comprises GM-CSF.
6. The method of claim 5 , wherein the composition further comprises IL 4.
7. The method of claim 1 , wherein the GM-CSF is present at a concentration of between about 200 U/ml to about 2000 U/ml.
8. The method of claim 1 , wherein the dendritic cells express high levels of MHC class molecules.
9. The method of claim 1 , wherein the dendritic cells have the capacity to stimulate resting T cells.