- Email: [email protected]
In vitro immunologic responsiveness of frozen stored human lymphocytes
ABSTRACTS-ELEVENTH ANNUAL MEETING
542 more insight granulocyte methods.
17. In
into unresolved freezing and
problems practical
such as selection
Vitro Immunologic Responsiveness of Frozen Stored Human Lymphocytes. D. M. STRONG, M. A. FACKTOR,” J. Wo~cly,* ASD K. W. SELL. (Naval Medical Research Institute, Bethesda, Maryland 20014).
Recent advances in transplantation have created a need for the use of immune competent frozen stored human lymphocytes (FSHL) in immunological testing. We have evaluated the immune responsiveness of FSHL in vitro by several criteria. Mononuclear cells (lo-20 X 10R) obtained from human peripheral blood by a standard FicollHypaque technique were suspended in 4°C media RPM1 1640 containing 10% fetal calf serum and 7.5% DMSO. Two milliliter aliquots were cooled at -l”C/min. in a Cryoson BV-4 programmed then -5”C/min. to freezing system to -3O”C, -80°C and stored in liquid nitrogen vapor. On the cell suspensions were thawed day of testing, rapidly in a 37°C water bath. Dimethylsulfoxide was diluted slowly out of the sample and cells resuspended in fresh RPM1 1640. It was dcmonstrated that FSHL possess surface immunoglobulins ( SJg) by immunofluorescence, form sheep erythrocyte ( E ) and sheep erythrocyte-antibodycomplement (EAC) rosettes comparable to fresh cells. In addition, the results show that FSHL respond to mitogens, specific antigens, as both stimulators and responders in the MLC reaction, and exhibit cell-mediated lymphocytotoxicity after in t&o sensitization. (Supported by Research Unit MR4318.01.0007ABG2 of the Bureau of Medicine and Surgery, U.S. Navy)
18. Cryopresewation of Human Grunulocytes wi:itk Hydroxyetkyl Starch. J. M. GORE,* S. M. HUNT,* W. A. CURBY,” AND F. J. LIONETTI. (Center for Blood Research, 800 Huntington Av., Boston, Massachusetts 02115, and Sias Laboratories of the Lahey Clinic Foundation, 211 Summit Avenue, Brookline, Massachusetts 02146). Granulocyte preservation was undertaken rising hydroxyethyl starch (HES) for hoth sedimentation an d cryopreservation of buffy coat white cells from CPD whole blood. Buffy coats were mixed with HES to a final concentration of 4” (w/v) and hematocrit of 30%, and sedimented in inverted plastic syringes at 20-22°C for 50-55 min. The leukocyte enriched ( 100-500X ) snpernatant was frozen at 2.O”C per min to -80°C (and
stored frozen up to 3 1110). Alternatively, setlimented leukocytes were frozen after a slow (2.0 ml/min ) addition of 10% dimethylsulfoxitle (DMSO) to 5%. Tnbcs were thawed with shaking for 4 minutes at 37”. Dimethylsulfoxide war removed by dilution with Hank’s solution containing CPD and centrifugation (800 A for 1.5 min ). The pellet of granulocytes was resuspended in Nornrosol (balanced electrolyte solution, Abbot Laboratories). Buffy coats centrifuged from 10 units yielded 65 -C 5%:. of the available whole blood leukocytes, of which 60 _f 5% were recovered after sedimentation in 4%’ HES. Freezing in 5% DMSO yielded 95 r+ 2% of the prefrozen leukocytes, while freezing with HES (4%) without DMSO reduced the lymphocytes to 20% of prefreeze levels but yielded 9076 of the granulocytes. Postthawed viability of granulocytes was estimated morphoIogically (Wright’s smears) and by their ability to inhibit for 100 min the rate of growth of E. coli. Complete inhibition was observed at a ratio of one E. coli to one granulocyte. Postthawed granulocytes were characterized by high myeloperoxidase activity and exclusion of trypan blue. Presently approximately 40% of the granulocytes in CPD whole blood can be recovered in a postthawed viable state. (Supported by ONR contract NOOO-14-73C-0100).
19. A
Lymphocytotoxicity “‘Cr-Re1ea.w Microussay for Cell-mediated Immunity to Cytomegalovirus Employing Cryopreserwtl Infected Target Cells. I’. H. THONG,* M. hf. VINCENT, S. A. HENSEN, D. A. FuccILLo,* w. A. STILES,' AND J. A. BELLANTI.* (Georgetown University School of Medicine, Washington, D.C., logical Associates, Bethescla, and NIH, Bethesda, Maryland).
MicrobioMaryland,
Existing methods for the measurement of cellmediated immunity (CMI) to virnses by lymphocytotoxicity “Cr-release techniques require the development of chronically infected cell lines to serve as target cells. The present study employed the WI-38 cell line infected with Cytomegalovirus (CMV) at a multiplicity of 1O:l. This potentially cytolytic infection was arrested by means of cryopreservation at a controlled rate of freezing: 1°C per min until -30°C: 10°C per min until -150°C: and immediate transfer to nitrogen vapor storage. The cryoprotective agent used was 7.5% DMSO. Both infected and control WI-38 cells were thawed rapidly on the morning of the experiment. The microassay procedure was performed as previously described (J. Immunol. 110, 1502, 1973). Specific “‘Cr immune release of
542 more insight granulocyte methods.
17. In
into unresolved freezing and
problems practical
such as selection
Vitro Immunologic Responsiveness of Frozen Stored Human Lymphocytes. D. M. STRONG, M. A. FACKTOR,” J. Wo~cly,* ASD K. W. SELL. (Naval Medical Research Institute, Bethesda, Maryland 20014).
Recent advances in transplantation have created a need for the use of immune competent frozen stored human lymphocytes (FSHL) in immunological testing. We have evaluated the immune responsiveness of FSHL in vitro by several criteria. Mononuclear cells (lo-20 X 10R) obtained from human peripheral blood by a standard FicollHypaque technique were suspended in 4°C media RPM1 1640 containing 10% fetal calf serum and 7.5% DMSO. Two milliliter aliquots were cooled at -l”C/min. in a Cryoson BV-4 programmed then -5”C/min. to freezing system to -3O”C, -80°C and stored in liquid nitrogen vapor. On the cell suspensions were thawed day of testing, rapidly in a 37°C water bath. Dimethylsulfoxide was diluted slowly out of the sample and cells resuspended in fresh RPM1 1640. It was dcmonstrated that FSHL possess surface immunoglobulins ( SJg) by immunofluorescence, form sheep erythrocyte ( E ) and sheep erythrocyte-antibodycomplement (EAC) rosettes comparable to fresh cells. In addition, the results show that FSHL respond to mitogens, specific antigens, as both stimulators and responders in the MLC reaction, and exhibit cell-mediated lymphocytotoxicity after in t&o sensitization. (Supported by Research Unit MR4318.01.0007ABG2 of the Bureau of Medicine and Surgery, U.S. Navy)
18. Cryopresewation of Human Grunulocytes wi:itk Hydroxyetkyl Starch. J. M. GORE,* S. M. HUNT,* W. A. CURBY,” AND F. J. LIONETTI. (Center for Blood Research, 800 Huntington Av., Boston, Massachusetts 02115, and Sias Laboratories of the Lahey Clinic Foundation, 211 Summit Avenue, Brookline, Massachusetts 02146). Granulocyte preservation was undertaken rising hydroxyethyl starch (HES) for hoth sedimentation an d cryopreservation of buffy coat white cells from CPD whole blood. Buffy coats were mixed with HES to a final concentration of 4” (w/v) and hematocrit of 30%, and sedimented in inverted plastic syringes at 20-22°C for 50-55 min. The leukocyte enriched ( 100-500X ) snpernatant was frozen at 2.O”C per min to -80°C (and
stored frozen up to 3 1110). Alternatively, setlimented leukocytes were frozen after a slow (2.0 ml/min ) addition of 10% dimethylsulfoxitle (DMSO) to 5%. Tnbcs were thawed with shaking for 4 minutes at 37”. Dimethylsulfoxide war removed by dilution with Hank’s solution containing CPD and centrifugation (800 A for 1.5 min ). The pellet of granulocytes was resuspended in Nornrosol (balanced electrolyte solution, Abbot Laboratories). Buffy coats centrifuged from 10 units yielded 65 -C 5%:. of the available whole blood leukocytes, of which 60 _f 5% were recovered after sedimentation in 4%’ HES. Freezing in 5% DMSO yielded 95 r+ 2% of the prefrozen leukocytes, while freezing with HES (4%) without DMSO reduced the lymphocytes to 20% of prefreeze levels but yielded 9076 of the granulocytes. Postthawed viability of granulocytes was estimated morphoIogically (Wright’s smears) and by their ability to inhibit for 100 min the rate of growth of E. coli. Complete inhibition was observed at a ratio of one E. coli to one granulocyte. Postthawed granulocytes were characterized by high myeloperoxidase activity and exclusion of trypan blue. Presently approximately 40% of the granulocytes in CPD whole blood can be recovered in a postthawed viable state. (Supported by ONR contract NOOO-14-73C-0100).
19. A
Lymphocytotoxicity “‘Cr-Re1ea.w Microussay for Cell-mediated Immunity to Cytomegalovirus Employing Cryopreserwtl Infected Target Cells. I’. H. THONG,* M. hf. VINCENT, S. A. HENSEN, D. A. FuccILLo,* w. A. STILES,' AND J. A. BELLANTI.* (Georgetown University School of Medicine, Washington, D.C., logical Associates, Bethescla, and NIH, Bethesda, Maryland).
MicrobioMaryland,
Existing methods for the measurement of cellmediated immunity (CMI) to virnses by lymphocytotoxicity “Cr-release techniques require the development of chronically infected cell lines to serve as target cells. The present study employed the WI-38 cell line infected with Cytomegalovirus (CMV) at a multiplicity of 1O:l. This potentially cytolytic infection was arrested by means of cryopreservation at a controlled rate of freezing: 1°C per min until -30°C: 10°C per min until -150°C: and immediate transfer to nitrogen vapor storage. The cryoprotective agent used was 7.5% DMSO. Both infected and control WI-38 cells were thawed rapidly on the morning of the experiment. The microassay procedure was performed as previously described (J. Immunol. 110, 1502, 1973). Specific “‘Cr immune release of