Method for producing ready to use, antigen-loaded or unloaded, cryoconserved mature dendritic cells
The invention relates to a method for producing ready to use, antigen loaded or unloaded, cryoconserved mature dendritic cells especially for the production of a vaccine containing said dendritic cells, wherein immature dendritic cells are cultivated in the presence of suitable maturation stimuli and the mature dendritic cells thus obtained are frozen. The dendritic cells can be loaded with antigen before freezing. The invention also relates to a vaccine which can be obtained according to the inventive method and to a composition containing frozen, mature dendritic cells which are loaded with antigen.
1. A method for preparation of ready-to-use cryoconserved mature dendritic cells, the method comprising:
(a) providing immature dendritic cells:
(b) culturing the immature dendritic cells in a culture medium containing a maturing cocktail comprising maturing stimulants IL-1β, IL-6, PGE 2 , and TNF-α to obtain mature dendritic cells, wherein said culture medium contains from 0.1 to 100 ng/ml IL-1β, from 0.1 to 100 ng/ml IL-6, from 0.1 to 1.0 μg/ml prostaglandin E 2 (PGE 2 ), and from 0.1 to 100 ng/ml TNF-α; and
(c) freezing the mature dendritic cells at a concentration of from 5×10 6 cells/ml to 20×10 6 cells/ml in a freezing medium comprising:
(i) autologous serum or autologous plasma;
(ii) 5-25% DMSO; and
(iii) 5-10% glucose,
to obtain frozen dendritic cells suitable after thawing for administration to a patient.
2. A method for preparation of ready-to-use cryoconserved mature dendritic cells, the method comprising:
(a) providing immature dendritic
(b) culturing the immature dendritic cells in a culture medium containing a maturing cocktail comprising IL-1, IL-6, prostaglandin E 2 (PGE 2 ) and TNFα to obtain mature dendritic cells: and
(c) freezing the mature dendritic cells at a concentration of from 5×10 6 cells/ml to 20×10 6 cells/ml in a freezing medium comprising:
(i) autologous serum or autologous plasma;
(ii) 5-25% DMSO; and
(iii) 5-10% glucose,
to obtain frozen dendritic cells suitable after thawing for administration to a patient.
3. A method for preparation of ready-to-use cryoconserved mature dendritic cells, the method comprising:
(a) providing immature dendritic cells:
(b) culturing the immature dendritic cells in a culture medium containing a maturing cocktail comprising interleukin 1β (IL-1β), interleukin 6 (IL-6), prostaglandin E 2 (PGE 2 ), and tumor necrosis factor α (TNFα) to obtain mature dendritic cells; and
(c) freezing the mature dendritic cells at a concentration of from 5×10 6 cells/ml to 20×10 6 cells/ml in a freezing medium comprising:
(i) autologous serum or autologous plasma;
(ii) 5-25% DMSO; and
(iii) 5-10% glucose,
to obtain frozen dendritic cells suitable after thawing for administration to a patient, wherein said dendritic cells are loaded with an antigen prior to said freezing, and wherein said antigen is a protein or a fragment thereof having at least 8 amino acids- and is added to the culture medium in a concentration of from 0.01 to 1000 μM.