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Dendritic cells are professional antigen presenting cells which are being used as adjuvants in tumor vaccination trials. They are generally grown in vitro from culture of blood monocytes, in presence of GM-CSF + IL-4 ± TNFa. Most clinical protocols currently include 4 to 10 weekly infusions of doses >106 cells, each inoculum coming from a simple culture of blood monocytes. In the present study, we have produced several millions of dendritic cells form a single leukapheresis: monocytes were isolated by elutriation (Beckman J ME) and then cultured in teflon bags in presence of 800 units/ml GM-CSF (Schering Plough) + 100 ng/ml IL-13 (Sanofi) + 10% fetal calf serum (FCS). The denditic cells from this single batch were aliquoted in many doses for potential multiple infusions and cryopreserved in 10% DMSO + 2% human albumin in Teflon-kapton Fresenius bags either at −1°C/min using a controlled rate freezer, or putting the bags directly in a −80°C mechanical freezer without controlling the temperature rate. Six experiments were carried out. After one month of cryopreservation, the cells were thawed in a 40°C water bath. Before and after freezing, cells were evaluated for immunophenotype (CD1a, CD14, CD40, CD80, CD83, CD86, CD54, CD58, CD16, CD32, CD64, HLA DR) and for their capacity to stimulate allogenic (MLR) or autologous (antigen presentation tests) lymphocytes. The results demonstrated that the mean recoveries after freezing in liquid nitrogen or at −80°C were respectively 67% ± 14 and 71% ± 13, without any significant difference between the two techniques. The immunophenotype was not modified by the freezing-thawing procedure, as well as the lymphocyte stimulating capacities. In conclusion, our study shows that substantial numbers of functional DCs can be derived from peripheral blood monocytes using teflon bags. DCs can be cryopreserved in a good laboratory practice setting for future clinical trials with an acceptable loss of cells and without modification of their functions. |
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