title: Controlled-rate freezer cryopreservation of highly concentrated peripheral blood mononuclear cells results in higher cell yields and superior autologous T-cell stimulation for dendritic cell-based immunotherapy.
abstract: Availability of large quantities of functionally effective dendritic cells (DC) represents one of the major challenges for immunotherapeutic trials against infectious or malignant diseases. Low numbers or insufficient T-cell activation of DC may result in premature termination of treatment and unsatisfying immune responses in clinical trials. Based on the notion that cryopreservation of monocytes is superior to cryopreservation of immature or mature DC in terms of resulting DC quantity and immuno-stimulatory capacity, we aimed to establish an optimized protocol for the cryopreservation of highly concentrated peripheral blood mononuclear cells (PBMC) for DC-based immunotherapy. Cryopreserved cell preparations were analyzed regarding quantitative recovery, viability, phenotype, and functional properties. In contrast to standard isopropyl alcohol (IPA) freezing, PBMC cryopreservation in an automated controlled-rate freezer (CRF) with subsequent thawing and differentiation resulted in significantly higher cell yields of immature and mature DC. Immature DC yields and total protein content after using CRF were comparable with results obtained with freshly prepared PBMC and exceeded results of standard IPA freezing by approximately 50 %. While differentiation markers, allogeneic T-cell stimulation, viability, and cytokine profiles were similar to DC from standard freezing procedures, DC generated from CRF-cryopreserved PBMC induced a significantly higher antigen-specific IFN-γ release from autologous effector T cells. In summary, automated controlled-rate freezing of highly concentrated PBMC represents an improved method for increasing DC yields and autologous T-cell stimulation.
authors.affiliation: Department of Dermatology, Venereology and Allergology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany. buhl@med.uni-goettingen.de
authors.forename: Timo
authors.initials: T
authors.lastname: Buhl
authors.forename: Tobias J
authors.initials: TJ
authors.lastname: Legler
authors.forename: Albert
authors.initials: A
authors.lastname: Rosenberger
authors.forename: Anke
authors.initials: A
authors.lastname: Schardt
authors.forename: Michael P
authors.initials: MP
authors.lastname: Schön
authors.forename: Holger A
authors.initials: HA
authors.lastname: Haenssle
mesh_terms: D019840:2-Propanol / Q000494:pharmacology; D001793:Blood Preservation / Q000379:methods*; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D003713:Dendritic Cells / Q000276:immunology*; D006801:Humans; D007167:Immunotherapy; D007937:Leukapheresis; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008213:Lymphocyte Activation; D013601:T-Lymphocytes / Q000276:immunology*; D014182:Transplantation, Autologous
chemical_list: D003451:Cryoprotective Agents; D019840:2-Propanol