Technical Report No. 17-85
Cryopreservation of human lymphocytes for use in immunologic tests: Report 2
Fujiwara S, Akiyama M, Yamakido M, Seyama T, Kobuke K, Hakoda M, Kyoizumi S, Jones SLEditor’s note: A publication based on this report was published in J Immunol Methods 90:265-73, 1986.
Summary
As the number of atomic bomb survivors has been decreasing each year, it would be advisable to preserve their valuable cells for future studies. Thus, cryopreservation of cells has become increasingly important.
The authors conducted experiments on cryopreservation of human lymphocytes and have previously reported the effects of cryopreservation on mitogen response and mixed lymphocyte culture. Having established a method of cryopreservation and evaluated its effects on lymphocyte subpopulation, immunoglobulin production, and cytotoxicity, the following conclusions were obtained.
1) The optimal conditions for cryopreservation were: the use of freezing and thawing media of pH 7.2, concentration of dimethyl sulfoxide of 10%, concentration of cells suspended in fetal calf serum at the time of freezing of from 5 to 15 x 106/ml, freezing rate of from -1 to -2 degrees centigrade/min, and thawing temperature at 37 degrees centigrade. Under such conditions, the average viability and recovery rate after cryopreservation were 90% and 80%, respectively.
2) Neither E receptor nor Fc receptor of the cell surface marker was affected by cryopreservation. The proportion of T cells, helper-inducer T cells, cytotoxic-suppressor T cells, B cells, monocytes, and natural killer (NK) cells was determined by monoclonal antibodies and was found to be stable following cryopreservation.
3) The ability of B lymphocytes to produce immunoglobulin was not significantly altered after cryopreservation.
4) Antibody-dependent cell-mediated crytotoxicity (ADCC) was not affected by cryopreservation when determined by a plaque assay using sheep red blood cells as target cells, but there was an average 35% reduction in ADCC after cryopreservation when examined by 3H-proline cytotoxicity assay with T-24 target cells.
5) NK cell activity showed an average of 40%-60% decrease immediately after thawing, but a recovery of NK cell activity was observed after preincubation for 18 hours. No change was observed in either lymphocyte subpopulations or viability after preincubation following cryopreservation, but as an increase was observed in the ratio of target cells binding to K-562, it was considered that preincubation may be a cause for the recovery of NK cell activity.
6) Freezing damages are considered to depend on the freezing process rather than on the length of cryopreservation period. This was confirmed by the fact that mitogen responses, E- and EAC-rosette formation, and NK cell activity could be kept intact for 12 months, 14 months, and 14 months, respectively.
As the number of atomic bomb survivors has been decreasing each year, it would be advisable to preserve their valuable cells for future studies. Thus, cryopreservation of cells has become increasingly important.
The authors conducted experiments on cryopreservation of human lymphocytes and have previously reported the effects of cryopreservation on mitogen response and mixed lymphocyte culture. Having established a method of cryopreservation and evaluated its effects on lymphocyte subpopulation, immunoglobulin production, and cytotoxicity, the following conclusions were obtained.
1) The optimal conditions for cryopreservation were: the use of freezing and thawing media of pH 7.2, concentration of dimethyl sulfoxide of 10%, concentration of cells suspended in fetal calf serum at the time of freezing of from 5 to 15 x 106/ml, freezing rate of from -1 to -2 degrees centigrade/min, and thawing temperature at 37 degrees centigrade. Under such conditions, the average viability and recovery rate after cryopreservation were 90% and 80%, respectively.
2) Neither E receptor nor Fc receptor of the cell surface marker was affected by cryopreservation. The proportion of T cells, helper-inducer T cells, cytotoxic-suppressor T cells, B cells, monocytes, and natural killer (NK) cells was determined by monoclonal antibodies and was found to be stable following cryopreservation.
3) The ability of B lymphocytes to produce immunoglobulin was not significantly altered after cryopreservation.
4) Antibody-dependent cell-mediated crytotoxicity (ADCC) was not affected by cryopreservation when determined by a plaque assay using sheep red blood cells as target cells, but there was an average 35% reduction in ADCC after cryopreservation when examined by 3H-proline cytotoxicity assay with T-24 target cells.
5) NK cell activity showed an average of 40%-60% decrease immediately after thawing, but a recovery of NK cell activity was observed after preincubation for 18 hours. No change was observed in either lymphocyte subpopulations or viability after preincubation following cryopreservation, but as an increase was observed in the ratio of target cells binding to K-562, it was considered that preincubation may be a cause for the recovery of NK cell activity.
6) Freezing damages are considered to depend on the freezing process rather than on the length of cryopreservation period. This was confirmed by the fact that mitogen responses, E- and EAC-rosette formation, and NK cell activity could be kept intact for 12 months, 14 months, and 14 months, respectively.