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Use of cryopreserved lymphocytes in mixed lymphocyte reaction: Comparison of different freezing protocols
ABSTRACTS-ELEVENTH
546
from patients with AML was 4.3 X 10” (range 0.7-9.5 x 100). Peripheral blood and bone marrow cells were collected into TC 199 with heparin and dimethylsuphoxide (final concentration 10%) was added as cryoprotective agent. Each collection was divided into a number of 2 ml glass vials or 2 ml screw-top plastic tubes and frozen at a controlled rate of 1°C per min to -60°C after which the rate of freezing was allowed to accelerate. The viability of CFC present in the stored peripheral blood and bone marrow collections was monitored at intervals by the use of the in citru agar colony culture method using normal human peripheral blood leucocytes as source of colony-stimulating factor. The results of these studies suggest that CFC and by implication haemopoietic stem cells obtained from patients with CGL and AML display good or perfect viability after storage in liquid nitrogen for periods of up to two years or more.
29. Use of Cryopreserved Lymphocytes in Mixed Lymphocyte Reuction: Comparison of Different Freezing Protocols. J. M. TURC (Laboratoire fusion
de Cryobiologie, Sanguine, Dijon
Centre (ZlOOO),
de TransFrance).
It is now well established that cryopreserved lymphocytes can be used in the mixed lymphocyte culture test. But the correlation of the results with those obtained by fresh cells is not always perfect and many technical parameters vary between laboratories. We have compared different freezing protocols and studied some parameters which may influence the recovery and functional capacities of lymphocytes after freezing and thawing. According to these results, it seems urgent to standardize a technique of freezing lymphocyte for immunological studies. SESSION 31.
D. CELLULAR
The Effect centration tion
CRYOBIOLOGY
Cooling Rate and DMSO Conon Low Temperature Preservnof Neonatal Rat Henrt Cells. G. M. of
c. vERHElZ,* AND F. G. J. (Department of Applied Physiology and Cryobiology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands ).
ALINK,* OFFERIJNS
c.
The preservation of rat temperature of liquid nitrogen, cryoprotectant, was described ( 1969) and by Janiszewski though in these publications
heart cells at the using DMSO as a by Offerijns et al. et al. ( 1972). Algood survival after
ANNUAL
MEETING
thawing was reported, optimal cooling rates and DMSO concentrations were not systematically investigated. We now present experiments on the influence of different cooling rates and DMSO concentrations on the survival of frozen rat heart cells. Suspensions of neonatal rat heart cells were prepared by trypsinization ( Hak et al. 1973 ). Cell suspensions in culture medium containing 2.5, 5, 7.5, or 10% DMSO were frozen at different cooling rates: 1, 5, 10, 30, and 50”Q’min from 0 to -100°C and then rapidly to -196°C. After thawing rapidly at +4O”C, cells were washed, plated in culture dishes and incubated at +37”C. Growth and contractility were studied after two and five days of culturing. Unfrozen-untreated, unfrozen-treated and frozen-treated cells were compared. On the second day, cells frozen at 5”C/min with 5% DMSO showed optimal survival of growth as well as contractility (73%). On the fifth day cells frozen at lO”C/min with 7.5% DMSO showed optimal survival of growth ( 100%). Survival of contractility remained optimal ( 75% ) with 57; DMSO and a cooling rate of 5”C/min. The effect of freezing and thawing on contraction frequency will be discussed. (Supported by the Foundation for Medical Research “FUNGO,” The Nctherlands). Hak, A. M., Offerijins, F. G. J., and Verheul, C. C. Cryobiclogy 10, 224-250 (1973). Janiszewski, E., and Wollenberger, A. Acta Biol. d4ed. Germ. 29, 135-147 (1972). Offerijns, F. G. J., Freud, G. E., and Krijnen, 1~. W. Nature (London) 222, 1174-1175 (1969). 32.
The Efiect of Cooling Rute and of DMSO on the Ultrastructure of Neonatal Rat Heurt Cells after Freezing and Thawing. G. M. J, AGTEHBEHC,* A. W. HELDEH,* G. J, OFFEAIJNS (Department of Applied Physiology and Cryobiology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands). ALINK," AND F.
The ultrastructure of dispersed neonatal rat heart cells was described by Kasten ( 1966). For embryonic chicken heart cells Wollenberger ( 1967) reported a higher proportion of damaged cells frozen with a cooling rate of l”C/min and 1 hf DMSO in comparison with unfrozen cells. Recently we observed differences in survival of neonatal rat heart cells frozen with different cooling rates and DMSO concentrations ( Alink et al. ). In this paper we present the results of an electron microscopical study on dispersed neonatal rat heart cells after freezing and thawing. We studied the
546
from patients with AML was 4.3 X 10” (range 0.7-9.5 x 100). Peripheral blood and bone marrow cells were collected into TC 199 with heparin and dimethylsuphoxide (final concentration 10%) was added as cryoprotective agent. Each collection was divided into a number of 2 ml glass vials or 2 ml screw-top plastic tubes and frozen at a controlled rate of 1°C per min to -60°C after which the rate of freezing was allowed to accelerate. The viability of CFC present in the stored peripheral blood and bone marrow collections was monitored at intervals by the use of the in citru agar colony culture method using normal human peripheral blood leucocytes as source of colony-stimulating factor. The results of these studies suggest that CFC and by implication haemopoietic stem cells obtained from patients with CGL and AML display good or perfect viability after storage in liquid nitrogen for periods of up to two years or more.
29. Use of Cryopreserved Lymphocytes in Mixed Lymphocyte Reuction: Comparison of Different Freezing Protocols. J. M. TURC (Laboratoire fusion
de Cryobiologie, Sanguine, Dijon
Centre (ZlOOO),
de TransFrance).
It is now well established that cryopreserved lymphocytes can be used in the mixed lymphocyte culture test. But the correlation of the results with those obtained by fresh cells is not always perfect and many technical parameters vary between laboratories. We have compared different freezing protocols and studied some parameters which may influence the recovery and functional capacities of lymphocytes after freezing and thawing. According to these results, it seems urgent to standardize a technique of freezing lymphocyte for immunological studies. SESSION 31.
D. CELLULAR
The Effect centration tion
CRYOBIOLOGY
Cooling Rate and DMSO Conon Low Temperature Preservnof Neonatal Rat Henrt Cells. G. M. of
c. vERHElZ,* AND F. G. J. (Department of Applied Physiology and Cryobiology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands ).
ALINK,* OFFERIJNS
c.
The preservation of rat temperature of liquid nitrogen, cryoprotectant, was described ( 1969) and by Janiszewski though in these publications
heart cells at the using DMSO as a by Offerijns et al. et al. ( 1972). Algood survival after
ANNUAL
MEETING
thawing was reported, optimal cooling rates and DMSO concentrations were not systematically investigated. We now present experiments on the influence of different cooling rates and DMSO concentrations on the survival of frozen rat heart cells. Suspensions of neonatal rat heart cells were prepared by trypsinization ( Hak et al. 1973 ). Cell suspensions in culture medium containing 2.5, 5, 7.5, or 10% DMSO were frozen at different cooling rates: 1, 5, 10, 30, and 50”Q’min from 0 to -100°C and then rapidly to -196°C. After thawing rapidly at +4O”C, cells were washed, plated in culture dishes and incubated at +37”C. Growth and contractility were studied after two and five days of culturing. Unfrozen-untreated, unfrozen-treated and frozen-treated cells were compared. On the second day, cells frozen at 5”C/min with 5% DMSO showed optimal survival of growth as well as contractility (73%). On the fifth day cells frozen at lO”C/min with 7.5% DMSO showed optimal survival of growth ( 100%). Survival of contractility remained optimal ( 75% ) with 57; DMSO and a cooling rate of 5”C/min. The effect of freezing and thawing on contraction frequency will be discussed. (Supported by the Foundation for Medical Research “FUNGO,” The Nctherlands). Hak, A. M., Offerijins, F. G. J., and Verheul, C. C. Cryobiclogy 10, 224-250 (1973). Janiszewski, E., and Wollenberger, A. Acta Biol. d4ed. Germ. 29, 135-147 (1972). Offerijns, F. G. J., Freud, G. E., and Krijnen, 1~. W. Nature (London) 222, 1174-1175 (1969). 32.
The Efiect of Cooling Rute and of DMSO on the Ultrastructure of Neonatal Rat Heurt Cells after Freezing and Thawing. G. M. J, AGTEHBEHC,* A. W. HELDEH,* G. J, OFFEAIJNS (Department of Applied Physiology and Cryobiology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands). ALINK," AND F.
The ultrastructure of dispersed neonatal rat heart cells was described by Kasten ( 1966). For embryonic chicken heart cells Wollenberger ( 1967) reported a higher proportion of damaged cells frozen with a cooling rate of l”C/min and 1 hf DMSO in comparison with unfrozen cells. Recently we observed differences in survival of neonatal rat heart cells frozen with different cooling rates and DMSO concentrations ( Alink et al. ). In this paper we present the results of an electron microscopical study on dispersed neonatal rat heart cells after freezing and thawing. We studied the