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In vitro generation of dendritic cells derived from cryopreserved CD34+ cells mobilized into peripheral blood in
M Enomoto1, H Nagayama, K Sato
1Department of Cell Processing, The Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedia, Minato-ku, Tokyo 108-8639, Japan.
Background:
Dendritic cells (DC) are APC that initiate primary T-cell dependent immune responses. They have been shown to be generated from CD34+ cells in BM, placental/umbilical cord blood (CB), and G-CSF mobilized peripheral blood CD34+ cells (PBSC). In recent clinical studies, DC were used as a vaccine for cancer patients and showed induction of their antitumor effects. Cryopreservation of CD34+ cells is important to extend the availability of cellular therapy with DC. However, little is known about the effect of cryopreservation on the functional maturation of DC.
Methods:
PBSC harvested from lymphoma patients mobilized with G-CSF and undergoing leukapheresis were cryopreserved at -135 degrees C for 3 days. Freshly isolated or cryopreserved PBSC were cultured with GM-CSF/SCF/tumor necrosis factor-alpha (TNF-alpha). After 14 days of culture, DC were harvested, washed, and used for phenotypical and functional analysis.
Results:
Cryopreserved PBSC, as well as freshly-isolated PBSC cultured for 14 days, gave rise to CD1a+ /CD4+ /CD11c+ /CD14low+ /CD25( -)/CD40+ / CD45RO+/CD80+/CD83+/CD86+/HLA-DR+ cells with dendritic morphology. DC derived from cryopreserved PBSC mobilized with G-CSF showed a similar endocytic capacity and chemotactic migratory capacities when compared with DC derived from freshly-isolated G-CSF mobilized PBSC. These DC also exhibited similar capacities in the primary allogeneic T-cell response.
Discussion:
These results indicate that cryopreserved G-CSF mobilized PBSC cultured with GM-CSF/SCF/TNF-alpha gave rise to DC that were morphologically, phenotypically and functionally similar to DC derived from fresh G-CSF mobilized PBSC. The observation indicates the clinical usefulness of cryopreserved CD34+ cells from lymphoma patients.