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Effect of cryoprotectant on granulocytes
ABSTRACTS, TSUJI (Transplantation ter, Tokai University Bohseidai, Isehara, Jaw
15TH
Immunology CenSchool of Medicine, Kanagawa 259-11,
1.
Membrane microviscosity is an important index expressing the various cell states. Our preliminary study showed that membrane microviscosity was changed in early stage of mixed lymphocyte cuItures. The utilization of cryopreservation of human peripheral lymphocytes is useful and convenient in the field of immunology. The purpose of this study is to investigate the possibility of utilizing cryopre.;ervation in the measurement of membrane microviscosity. Human peripheral lymphocytes were preserved in 10% dimethyl sulfoxide (DMSO) and 20% AB serum-supplemented RPMI-1640 at -80°C. At an appropriate time after cryopreservation, the preserved lymphocytes were thawed and washed three times with phosphate-bu#ered saline (PBS). It is well known that the microviscosity is indicated as a function of fluorescence polarization (P-value), and membrane microviscosity is represented by measuring the P-value of cells whose membranes are labeled with a fluorescent probe. The fresh or cryopreserved lymphocytes were labeled with I X IO-” M dinitrophenyl hexatriene (DPH) at room temperature for 60 min. P-values of 5 X IO’-DPH labeled lymphocytes were measured with a microviscosimeter, MV-1, Elscint, Israel, at 25°C. When cell viability was more than 90%, almost no change of P-values was observed in comparing fresh and cryopreserved lymphocytes from the same source (1, 2, or 4 weeks). P-values of fresh lymphocytes and cryopreserved lymphocytes (4 weeks) were 255 t 3 and 256 * 4, respectively. When cell viability was reduced markedly, significant changes in P-values were observed. As the result, P-values of lymphocytes which were cryopreserved in good condition were almost the same as those of fresh lymphocytes. It will be expected that the cryopreserved lymphocytes are utilized in the measurement of membrane microviscosity which express a delicate membrane function and a physioIogLca1 membrane state.
51. E&zt of Cryoprotectant on G+anulocyte,s T. YA.MASHITA,~ N. IMAIZUhfI,f ANll S. YUASA $ ( fLaboratory of Physiological Chemistry and $ Blood Transfusion Service, School of Medicine, Juntendo University, Tokyo 113, Japan). The methyl glycol
effect of cryoprotective sulforide ( DMSD), upon the function
agents such as diglycerol, and ethylene of polymorphonuclear
ANNUAL
MEETING
697
nentrophils (PMlNs) during the storage at a low temperature was investigated. Increasing concentrations of each cryoprotectant caused an increasing inhibition of chemotaxis with the complete inhibition at 16.7% where most of PMNs were still viable. This inhibition was eliminated by the removal of the cryoprotectant by washing. As for exposure of PMNs to a lower concentration of the agent for 1 hr, it resulted in nearly the same chemotaxis as the contro1 although the recovery of chemotaxis was not seen at 16.7%. With 20”hr exposure, the inhibitory effect on chemotaxis was removed by washing when a 4.2% agent was used, but using an 8.3% agent, chemotaxis was not restored; PMNs exposed to DMSO displayed almost the same chemotaxis as a control. With 16.7% agent cryoprotectanttreated PMNs, damage of a large number of cells was observed besides the complete inhibition of chemotaxis. The ability of PMNs to ingest bacteria was not markedly inhibited by a I-hr exposure although complete inhibition was ohserved using 16.7% ethylene glycol. As for 20-hr exposure, ingestive ability remained in all cases using a 4.2% concentration. Using an 8.3% concentration, the DMSO-exposed PMNs retained a good ingestive ability. 52.
HLA-D Typing of Human Lymphocfles Using Frozen and Thawed Spermatozoa. K. HALIM,* H. FESTENSTEIN,' AXD J. FARRANT (The London Hospital Medical College, London El and The MRC Clinical Research Centre, Harrow, United Kingdom )
A method is described for the preservation in liquid nitrogen of “hagloid” populations of human spermatozoa prepared before freezing by treatment with anti-HLA antisera. The freezing technique employed gIycero1 (7.5%, v/v) and heatinactivated AB serum (5%, v/v) with a two-step cooling method of 5 min at -40°C before storage at -196°C. The thawed spermatozoa were used as stimulators of lymphocytes in microplate culture with measurement of the incorporation of LSH]thymidine into DNA. The typing results obtained when using fresh spermatozoa and also with other cellular typing methods. 53.
Freeze Preservation of Human Platelets for Tramfusion at -196°C. S. SUMIDA (National Fukuoka Central Hospital Jonai 2-2, Fukuoka 810, Japan ) ,
Blood unteers
(200 ml) into blood
was drawn from bags containing
healthy CPD
volsolu-
15TH
Immunology CenSchool of Medicine, Kanagawa 259-11,
1.
Membrane microviscosity is an important index expressing the various cell states. Our preliminary study showed that membrane microviscosity was changed in early stage of mixed lymphocyte cuItures. The utilization of cryopreservation of human peripheral lymphocytes is useful and convenient in the field of immunology. The purpose of this study is to investigate the possibility of utilizing cryopre.;ervation in the measurement of membrane microviscosity. Human peripheral lymphocytes were preserved in 10% dimethyl sulfoxide (DMSO) and 20% AB serum-supplemented RPMI-1640 at -80°C. At an appropriate time after cryopreservation, the preserved lymphocytes were thawed and washed three times with phosphate-bu#ered saline (PBS). It is well known that the microviscosity is indicated as a function of fluorescence polarization (P-value), and membrane microviscosity is represented by measuring the P-value of cells whose membranes are labeled with a fluorescent probe. The fresh or cryopreserved lymphocytes were labeled with I X IO-” M dinitrophenyl hexatriene (DPH) at room temperature for 60 min. P-values of 5 X IO’-DPH labeled lymphocytes were measured with a microviscosimeter, MV-1, Elscint, Israel, at 25°C. When cell viability was more than 90%, almost no change of P-values was observed in comparing fresh and cryopreserved lymphocytes from the same source (1, 2, or 4 weeks). P-values of fresh lymphocytes and cryopreserved lymphocytes (4 weeks) were 255 t 3 and 256 * 4, respectively. When cell viability was reduced markedly, significant changes in P-values were observed. As the result, P-values of lymphocytes which were cryopreserved in good condition were almost the same as those of fresh lymphocytes. It will be expected that the cryopreserved lymphocytes are utilized in the measurement of membrane microviscosity which express a delicate membrane function and a physioIogLca1 membrane state.
51. E&zt of Cryoprotectant on G+anulocyte,s T. YA.MASHITA,~ N. IMAIZUhfI,f ANll S. YUASA $ ( fLaboratory of Physiological Chemistry and $ Blood Transfusion Service, School of Medicine, Juntendo University, Tokyo 113, Japan). The methyl glycol
effect of cryoprotective sulforide ( DMSD), upon the function
agents such as diglycerol, and ethylene of polymorphonuclear
ANNUAL
MEETING
697
nentrophils (PMlNs) during the storage at a low temperature was investigated. Increasing concentrations of each cryoprotectant caused an increasing inhibition of chemotaxis with the complete inhibition at 16.7% where most of PMNs were still viable. This inhibition was eliminated by the removal of the cryoprotectant by washing. As for exposure of PMNs to a lower concentration of the agent for 1 hr, it resulted in nearly the same chemotaxis as the contro1 although the recovery of chemotaxis was not seen at 16.7%. With 20”hr exposure, the inhibitory effect on chemotaxis was removed by washing when a 4.2% agent was used, but using an 8.3% agent, chemotaxis was not restored; PMNs exposed to DMSO displayed almost the same chemotaxis as a control. With 16.7% agent cryoprotectanttreated PMNs, damage of a large number of cells was observed besides the complete inhibition of chemotaxis. The ability of PMNs to ingest bacteria was not markedly inhibited by a I-hr exposure although complete inhibition was ohserved using 16.7% ethylene glycol. As for 20-hr exposure, ingestive ability remained in all cases using a 4.2% concentration. Using an 8.3% concentration, the DMSO-exposed PMNs retained a good ingestive ability. 52.
HLA-D Typing of Human Lymphocfles Using Frozen and Thawed Spermatozoa. K. HALIM,* H. FESTENSTEIN,' AXD J. FARRANT (The London Hospital Medical College, London El and The MRC Clinical Research Centre, Harrow, United Kingdom )
A method is described for the preservation in liquid nitrogen of “hagloid” populations of human spermatozoa prepared before freezing by treatment with anti-HLA antisera. The freezing technique employed gIycero1 (7.5%, v/v) and heatinactivated AB serum (5%, v/v) with a two-step cooling method of 5 min at -40°C before storage at -196°C. The thawed spermatozoa were used as stimulators of lymphocytes in microplate culture with measurement of the incorporation of LSH]thymidine into DNA. The typing results obtained when using fresh spermatozoa and also with other cellular typing methods. 53.
Freeze Preservation of Human Platelets for Tramfusion at -196°C. S. SUMIDA (National Fukuoka Central Hospital Jonai 2-2, Fukuoka 810, Japan ) ,
Blood unteers
(200 ml) into blood
was drawn from bags containing
healthy CPD
volsolu-