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.

Biospecimen Science of Blood for Peripheral Blood Mononuclear Cell (PBMC) Functional Applications

“…The progressive rate freezing program may be critical for sensitive cell types: use of a controlled rate freezer leads to higher yields of dendritic cells and to higher autologous T cell stimulation [67] than freezing with isopropanol. The long-term storage time and temperature of the frozen cells is another critical factor.…”

Method Validation for Automated Isolation of Viable Peripheral Blood Mononuclear Cells

“…Buhl et al compared CRF freezing with standard isopropyl alcohol freezing for downstream dendritic cell applications. 32 They showed that while similar dendritic cell viability was seen with the two methods, induction of antigen-specific IFN-g release from autologous effector T cells was significantly higher with CRF. In our study, the two freezing methods, Mr. Frosty and Controlled Rate Freezer, gave largely similar cell recovery, viability, cell subpopulation distribution, and susceptibility to EBV immortalization.…”

Internalization routes of cell‐penetrating melanoma antigen peptides into human dendritic cells

“…The Melan‐A‐TAT fusion peptide [CELAGIGILTVRKKRRQRRR, Melan‐A sequence underlined] was synthesized using standard fluorenylmethoxycarbonyl (Fmoc) chemistry. The N‐terminal Cys residue was added to enable coupling of Alexa Fluor 488 (Alexa488) C 5 maleimide (Invitrogen, Carlsbad, CA, USA).…”