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Published on: August 1, 2013
A cryopreservation method of human peripheral blood mononuclear cells for efficient production of dendritic cells
Insights
Cryopreservation of peripheral blood mononuclear cells (PBMC) using 12% dimethylsulfoxide (DMSO) and fetal calf serum maintains high viability for dendritic cells (DC). This method ensures efficient recovery and function of cryopreserved dendritic cells for immunological studies.
Area of Science:
- Immunology
- Cell Biology
- Cryobiology
Background:
- Cryopreservation of fresh peripheral blood mononuclear cells (PBMC) is crucial for immunological research.
- Dendritic cells (DC), potent antigen-presenting cells, are highly sensitive to cryopreservation procedures.
- Existing methods may not optimally preserve DC viability and function.
Purpose of the Study:
- To establish an effective cryopreservation method for unstimulated fresh PBMC, focusing on dendritic cell (DC) preservation.
- To evaluate the recovery, differentiation, and function of cryopreserved DC and DC precursors.
- To compare the efficacy of different cryoprotective solutions for PBMC and DC cryopreservation.
Main Methods:
- PBMC were cryopreserved using varying concentrations of dimethylsulfoxide (DMSO) and fetal calf serum.
- Recombinant (r) GM-CSF and rIL-4 were used for DC recovery and differentiation.
- Cryopreserved PBMC and derived DC were assessed for viability, antigen expression (HLA-DR, HLA-DQ, CD80, CD86), and allogeneic stimulation capacity.
Main Results:
- PBMC cryopreserved with 12% DMSO and 25-30% fetal calf serum yielded efficient DC recovery and differentiation.
- Established DC cryopreserved with 12% DMSO maintained >90% viability.
- The 12% DMSO solution was superior to 10% DMSO and a previously reported DC freezing medium.
- DC from cryopreserved PBMC expressed key activation markers and stimulated allogeneic PBMC comparably to fresh PBMC.
Conclusions:
- A cryopreservation method using 12% DMSO and fetal calf serum enables safe and effective preservation of DC and DC precursors within PBMC.
- This method supports high viability and functional recovery of dendritic cells, facilitating their use in immunological experiments.
- The established protocol offers a reliable approach for cryopreserving essential components of the immune system for research purposes.
Abstract:
The establishment of a cryopreservation method for unstimulated fresh peripheral blood mononuclear cells (PBMC) with nearly 100% viability would greatly contribute to the conduct of various immunological experiments. The cells most sensitive to freezing and thawing procedure seem to be dendritic cells (DC) and their precursors, which are of the most potent antigen-presenting cells. The authors investigated and established a method of cryopreserving fresh PBMC from which DC were recovered and differentiated efficiently by using recombinant (r) GM-CSF and rIL-4. PBMC frozen in the presence of 12% dimethylsulfoxide and 25-30% fetal calf serum recovered DC as efficiently as freshly obtained PBMC. Established DC could also be cryopreserved in the presence of 12% DMSO with their viability maintained at more than 90%. The 12% DMSO freezing solutions were superior to both the 10% DMSO solution and the previously reported DC freezing medium (2 M or 15.4% DMSO). The DC obtained from the cryopreserved PBMC expressed HLA-DR, HLA-DQ, CD80 and CD86 antigens, and stimulated allogenic PBMC to an extent almost identical to that obtained from fresh PBMC. These findings indicate that the conditioned medium utilized here enables safe cryopreservation of DC and DC precursors in PBMC.
