A simple cryopreservation method for dendritic cells and cells used in their derivation and functional assessment.
BACKGROUND
Cryopreservation and storage permitting multiple treatments with single donations is of practical importance to cellular therapies. HES and DMSO, used successfully in simple clinical procedures for freezing marrow and peripheral blood progenitor cells at -80 degrees C, was tested on antigen-presenting dendritic cells (DCs) and cells used in their derivation.


METHODS
DCs cultured in serum-free media from adherent or CD14+ apheresis MNCs (n = 36) in the presence of GM-CSF + IL4 +/- TNFalpha were frozen and stored at -80 degrees C in 6-percent HES, 5-percent DMSO, and 4-percent HSA. Apheresis MNCs, CD14+ monocytes, and lymphocytes were similarly frozen and later thawed for culture. Cells were assayed for viability, DC phenotype, mixed lymphocyte reaction, and antigen presentation before and 3, 6, 9, 12 or more months after freezing.


RESULTS
DCs retained viability (82 +/- 2.3%) for at least 24 months. Mature and immature phenotype and function were preserved. Thawed MNCs and CD14+ cells differentiated to DCs and lymphocytes maintained high functional viability (92 +/- 3%) comparable to prefreeze levels.


CONCLUSIONS
A simple -80 degrees C freezing and storage method that combines extracellular (HES) and intracellular (DMSO) agents is practical and preserves functional viability of DCs, MNCs, CD14+ monocytes, and lymphocytes.
Celluzzi
Christina M
CM
Blood and Cell Therapy Development, American Red Cross Biomedical Research and Development, Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855, USA. celluzzi@usa.redcross.org
Welbon
Craig
C
D001781:Blood Component Removal; D017252:CD3 Complex / Q000032:analysis; D002452:Cell Count; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D016895:Culture Media, Serum-Free; D003713:Dendritic Cells / Q000166:cytology / Q000502:physiology*; D004121:Dimethyl Sulfoxide; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D006895:Hydroxyethyl Starch Derivatives; D015847:Interleukin-4 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D008214:Lymphocytes / Q000276:immunology / Q000502:physiology; D009000:Monocytes / Q000276:immunology / Q000502:physiology; D012709:Serum Albumin; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017252:CD3 Complex; D003451:Cryoprotective Agents; D016895:Culture Media, Serum-Free; D006895:Hydroxyethyl Starch Derivatives; D018950:Lipopolysaccharide Receptors; D012709:Serum Albumin; D014409:Tumor Necrosis Factor-alpha; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004121:Dimethyl Sulfoxide