Freezing and thawing of bone marrow-derived murine dendritic cells with subsequent retention of immunophenotype and of antigen processing and presentation characteristics.
Murine dendritic cells (DCs) are widely used for experimental vaccinations in mouse models. A high-yield method for freezing and thawing batches of these cells, if compatible with retention of cell immunophenotype, would reduce the time required for repeated preparations from DC precursors in bone marrow (BM), as well as variability among lots. Following depletion of specific lineages, murine bone marrow cells from C57BL/6 inbred-strain mice were grown in medium containing 10% fetal calf serum (FCS) and granulocyte/macrophage colony-stimulating factor (GM-CSF); after 6 days, large numbers of immature DCs were obtained. The immature cells were frozen in complete medium with GM-CSF and 10% DMSO, at a cell density of 5x10(6) DCs/ml. After thawing, 80% of DCs survived; they were induced to mature by addition of lipopolysaccharide (LPS). In comparison with fresh DCs, the thawed DCs had similar morphology, purity, and expression of class I (H-2D(b) and H-2K(b)) and class II major histocompatibility complex (MHC) proteins, as well as CD11b, CD11c, CD40, CD80, and CD86 molecules. Freeze-thawing did not affect trafficking to T cell areas of spleen, nor reduce the capacity to stimulate an alloresponse. Frozen-thawed cells were also proficient at uptake, processing, and presentation of native or denatured ovalbumin (OVA) protein to a peptide-specific T cell hybridoma, and were able to induce T cell responses in vivo after being loaded with denatured OVA protein. The ability to freeze and thaw DCs, and to obtain high yields without altering their essential properties, will facilitate future immunotherapy experiments in laboratory mouse models.
Sai
Tao
T
Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA.
Milling
Simon W F
SW
Mintz
Beatrice
B
D000818:Animals; D017951:Antigen Presentation; D001854:Bone Marrow Cells / Q000166:cytology* / Q000276:immunology* / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002465:Cell Movement / Q000276:immunology; D002470:Cell Survival / Q000276:immunology; D002478:Cells, Cultured; D003470:Culture Media; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology* / Q000378:metabolism / Q000637:transplantation; D018984:Epitopes, T-Lymphocyte / Q000276:immunology / Q000378:metabolism; D005260:Female; D005312:Fetal Blood; D005615:Freezing; D006825:Hybridomas; D016130:Immunophenotyping; D007275:Injections, Intravenous; D007959:Lymphocyte Culture Test, Mixed / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D010047:Ovalbumin / Q000276:immunology / Q000378:metabolism; D011489:Protein Denaturation; D013154:Spleen / Q000166:cytology / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology
D003470:Culture Media; D018984:Epitopes, T-Lymphocyte; D010047:Ovalbumin