Bulk cryopreservation of lymphocytes in glycerol.
The authors describe a method for freezing large amounts of peripheral blood lymphocytes (PBL) in a 20 percent glycerol solution. Between 0.6 and 4.3 X 10(9) cells in autologous plasma were frozen in polyethylene freezing bags in a final volume of 50 ml. The recovery after thawing averaged 89 +/- 14 percent with a mean viability by trypan blue dye exclusion of 80 +/- 7 percent (n = 11). In aliquots of fresh and frozen-thawed PBL from the same subjects radiolabeled with 111In, the radiolabeling efficiency for both fresh and thawed cells was 49 +/- 15 percent (p = 0.98, n = 5). The mitogen mean stimulation indices for glycerol-frozen cells (471 with phytohemagglutinin-M, 176 with pokeweed mitogen, and 380 with concanavalin A) were superior to those of cells frozen by a standard technique with dimethylsulfoxide (DMSO) (141, 47, and 123; p less than 0.05) and comparable to those of fresh PBL (403, 75, and 147). In a mixed lymphocyte culture, glycerol-frozen PBL showed significantly greater responsiveness to a pool of stimulator cells than did PBL frozen in DMSO (p = 0.03). Thawed cells are viable and functional as demonstrated by their response to mitogens and their ability to stimulate and respond in mixed lymphocyte culture.
Areman
E M
EM
Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland.
Simonis
T B
TB
Carter
C S
CS
Read
E J
EJ
Klein
H G
HG
D001793:Blood Preservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide; D005260:Female; D005615:Freezing; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D007958:Leukocyte Count; D008213:Lymphocyte Activation / Q000187:drug effects; D007959:Lymphocyte Culture Test, Mixed; D008214:Lymphocytes / Q000187:drug effects* / Q000276:immunology; D008297:Male
D003451:Cryoprotective Agents; D005990:Glycerol; D004121:Dimethyl Sulfoxide