In Vitro Production of Dendritic Cells from Human Blood Monocytes for Therapeutic Use

Abstract

Dendritic cells (DC) are professional antigen-presenting cells that are promising adjuvants for clinical immunotherapy. Methods to generate in vitro large numbers of functional human DC using either peripheral blood monocytes or CD34(+) pluripotent hematopoietic progenitor cells have been now developed. For this purpose, their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. In the present review, we give our experience of such a procedure: it includes collection of mononuclear cells by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + human serum (autologous patient's serum or AB serum) or in a serum-free medium (AIM V). The characteristics of monocyte-derived DC grown in these various conditions varied mainly regarding their phenotype and their morphology in confocal microscopy, whereas no significant differences were found in their capacity to phagocytize latex particles and to stimulate allogeneic (MLR) or autologous lymphocytes (antigen-presentation tests). The DC were also cryopreserved in bags (either by putting the bags directly in a -80 degrees C mechanical freezer or using a classical liquid nitrogen controlled-rate freezer at -1 degrees C/min) in a solution containing 10% dimethyl sulfoxide (Me(2)SO) and 2% human albumin in doses of DC available for several infusions. The mean recoveries after freezing and thawing were not statistically different (around 70%). The immunophenotype of DC, as well as the T lymphocyte-stimulating capacity, were not modified by the freezing--thawing procedure. The results obtained demonstrate that the experimental conditions we set up are easily applicable in clinical trials and lead to large numbers of well-defined DC. Clinical trials using DC already published will be discussed.

Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells

“…21,42 CD40-activated B cells have an important advantage for this purpose in that they can be readily expanded in vitro to a relatively large numbers (Figure 1), whereas, in contrast, monocytes differentiating in vitro into DCs do not undergo cell division. 22 Cryopreserved CD40-activated B cells also retain their APC function on thawing and are relatively cost-effective to produce. 26,27 In addition, because B cells stimulated with t-CD40-L cells or recombinant sCD40-L were equally effective at generating alloantigen-specific Treg, the use of sCD40-L may significantly improve the clinical applicability of the procedure.…”

“…For example, DCs are relatively rare in peripheral blood and are usually derived from apheresis or marrow sources, including monocytes. 22,23 Further, DCs are not homogeneous and include multiple subsets with different functional capacities. 24 Finally, there is no effective way to expand human The online version of this article contains a data supplement.…”

Uptake of Apoptotic K562 Leukaemia Cells by Immature Dendritic Cells is Greatly Facilitated by Serum

“…Others have studied the importance of AS for the generation of optimally immunostimulatory DCs. Garderet et al [22] found significantly higher expression of CD40, CD54 and CD86 on immature DCs generated in RPMI-1640 with 10% AS than in DCs generated in SFM. This difference was augmented after maturation for 2 days.…”

The large scale generation of dendritic cells for the immunization of patients with non-small cell lung cancer (NSCLC)

“…CD14+ cells, non-proliferative DC precursors, are obtained from leukapheresis procedures, buffy coat preparations, or peripheral blood draws [21][22][23]. In the laboratory, CD14+ monocytes are purified from blood using elutriation procedures [24,25], immunomagnetic purification [7] or adherence [26,27] steps. In some cases, DCs are derived from a proliferative CD34+ cell population and are obtained from the peripheral blood following mobilization using G-CSF (granulocyte colony stimulating factor) [28] and/or GMCSF (granulocyte monocyte colony stimulating factor) infusions [28][29][30].…”