Method for in vitro proliferation of dendritic cell precursors and their use to produce immunogens
View Patent ↗A method for producing proliferating cultures of dendritic cell precursors is provided. Also provided is a method for producing mature dendritic cells in culture from the proliferating dendritic cell precursors. The cultures of mature dendritic cells provide an effective means of producing novel T cell dependent antigens comprised of dendritic cell modified antigens or dendritic cells pulsed with antigen, including particulates, which antigen is processed and expressed on the antigen-activated dendritic cell. The novel antigens of the invention may be used as immunogens for vaccines or for the treatment of disease. These antigens may also be used to treat autoimmune diseases such as juvenile diabetes and multiple sclerosis.
1. A method of producing a population of dendritic cells comprising the steps of:
(a) providing a tissue source comprising dendritic cell precursors;
(b) treating the tissue source to enrich the proportion of dendritic precursor cells;
(c) culturing cells obtained from treatment of the tissue source in medium comprising GM-CSF at a concentration of about 400-800 U/ml and IL-4 at a concentration of about 500-1000 U/ml for a period of time sufficient to produce dendritic cells.
2. The method according to claim 1 , wherein the tissue source is blood.
3. The method according to claim 2 , wherein the blood is cord blood.
4. The method according to claim 1 , wherein the tissue source is human.
5. The method according to claim 1 , wherein the tissue source comprises mononuclear cells.
6. The method according to claim 5 , wherein said mononuclear cells include monocytes.
7. The method of claim 2 , wherein step (b) comprises obtaining a population of leukocytes substantially free of platelets and red blood cells.
8. The method of claim 1 , wherein step (b) comprises removing the majority of nonadherent, newly-formed granulocytes from the cultures by gentle washes.
9. The method of claim 1 , wherein step (b) comprises removing undesirable cells by panning using a plastic surface.
10. A method of producing a population of 3-8×10 6 dendritic cells comprising the steps of:
(a) providing a tissue source which is blood;
(b) treating the blood to enrich the proportion of dendritic precursor cells;
(c) culturing cells obtained from treatment of the tissue source in medium comprising GM-CSF at a concentration of about 400-800 U/ml and IL-4 at a concentration of about 500-1000 U/ml for a period of time sufficient to produce dendritic cells;
whereby a population of 3-8×10 6 dendritic cells is produced from 40 ml of blood.
11. A method of producing a population of dendritic cells comprising the steps of
(a) providing a tissue source comprising dendritic cell precursors;
(b) treating the tissue source to enrich the proportion of dendritic precursor cells;
(c) culturing cells obtained from treatment of the tissue source in medium comprising GM-CSF and IL-13 for a period of time sufficient to produce dendritic cells.