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Cryopreservation of schistosoma mansoni schistosomula
ABSTRACTS,
586
16th ANNUAL
determined spectrophotometrically at 540 nm. The recovery of red blood cells with 20% glycerol in 3% trisodium citrate without addition of antifreeze was 100%. However, addition of 0.5% of both high molecular weight glycopeptide (lO,SOO-23,000) and peptide antifreeze compounds (5000) produced 95% hemolysis in red blood cells in the presence of glycerol. The low molecular weight glycopeptide compounds (3000) cause the same amount of hemolysis at a concentration of 4.0% w/v). Both the glycopeptide and peptide antifreezes cause ice growth along the c’axis, the nonpreferred axis of growth. The resulting spicular ice growth most likely produces shearing forces leading to disruption of the red blood cell membrane which would explain the absence of cryoprotection in the presence of glycerol and polyvinylpyrrolidone. 12. Cold Hardiness and Adaptive Potential of an Anlarctic Insect. J. G. BAUST (Department of
Biology, University Texas 77004).
of Houston,
Houston,
Befgica antarctica, a wingless fly and the only freeliving holometabolous insect of the Antarctic continent, is uniquely adapted for survival under perennial “winter conditions.” Larvae are freezing tolerant throughout the year and elaborate a complex of cryoprotectants including glycerol, erythritol, glucose, sucrose, and trehalose. Maintenance on artificial diets indicates that “antifreeze” profiles have foodsource and temperature-dependent components. Direct utilization of dietary cryoprotectants is suggested. The terrestrial microhabitat of the mid-Antarctic Peninsula provides a novel thermostable environment. The annual range of habitat temperatures did not exceed -7 to 11°C during the 2-year study. Accordingly this species lacks adaptive plasticity common to arctic insects. The following indicators of chill tolerance did not vary seasonally or during acclimation attempts. Supercooling points remained constant at -5.7 5 O.S”C. Lower lethal temperatures were also constant at -10°C (LT 50), and prolonged exposure to 310°C resulted in impaired carbohydrate metabolism. The thermostability of the environment of this species has resulted in the evolution of an obligate insect homeotherm.
13. Cryopreser\*afion
of
Sc,histosoma
Mansoni
E. R. JAMES (London School of Hygiene and Tropical Medicine, Winches Farm Field Station, 395 Hatfield Road, St. Albans, Hertfordshire AL4 OXQ, United Kingdom).
Schistosomula.
Schistosomula obtained by in vitro transformation of cercariae have been cryopreserved successfully only by using a two-step cooling schedule. During the first cooling step nucleation must occur at or close to the
MEETING
freezing point of the suspending medium, approximately - 10.5”C, since supercooling by even 1.5”C before nucleation leads to an eight-fold drop in survival. Below the freezing point survival declines progressively as the temperature is lowered, being lost completely by -40°C. However, samples cooled rapidly to - 196°C from intermediate temperatures below about -20°C yield some viable organisms on thawing. The best intermediate temperature depends chiefly on the cooling rate of the first step and on the type and concentration of cryoprotectant; the faster the cooling rate the lower the intermediate temperature required for peak survival. The optimum combination appears to be 1°C min-’ to -30°C using 4.56 M methanol. With 2.3 M ethanediol, the peak is lower and occurs at -20°C. The second cooling step is required to be as fast as possible, approximately 10,OOO”Cmin-’ in these studies. Rapid warming, at approximately 10,OOO”C min-‘, coupled with concomitant dilution to remove the cryoprotectant is necessary to achieve any survival. These results suggest that avoidance of intracellular ice formation during the first cooling step is essential; that the number of undamaged schistosomula is depleted during the slow cooling step probably as a result of osmotic stress and/or salt and cryoprotectant toxicity, as well as intracellular ice formation; that intracellular ice is induced in all organisms during the second cooling step: and that rapid warming is necessary to minimise the damage due to this intracellular ice. The post-in vitro transformation age is important also, peak survival occurring with organisms 60 to 90 min old: this phenomenon probably results from the complex physiological and morphological changes which accompany the transition from a free-living (cercaria) to a parasitic (schistosomulum) existence. With all the conditions optimal, approximately 30% of cryopreserved schistosomula show normal motility and following injection into mice 6.1% develop to adult worms (compared to approximately 30% for unfrozen organisms), indicating approximately 20% are infective. SESSION 14. Supercooling organic
2. CELL
BIOLOGY
und Supersafuration Salt Solutions.
ofAqueous
In-
A. P. MACKENZIE (Center for Bioengineering RF-52, University of Washington, Seattle, Washington 98195).
Previous studies (MacKenzie, A. P., Cryobiology 14, 705-706, 1977) showed that dilute aqueous NaCl solutions readily supercooled lo- 15°C and that eutectic crystallization was not usually seen above -35°C. Since totally solidified aqueous NaCl underwent an equilibrium eutectic melting at -2l”C, it was clear that NaCl supersaturated in practice in the presence of ice. Supercoolings of IO to 15°C suggested heterogeneous nucleation of ice. Whether or not the nucleation of the NaCI was heterogeneous or homogeneous was not determined. We now report experiments in which the
586
16th ANNUAL
determined spectrophotometrically at 540 nm. The recovery of red blood cells with 20% glycerol in 3% trisodium citrate without addition of antifreeze was 100%. However, addition of 0.5% of both high molecular weight glycopeptide (lO,SOO-23,000) and peptide antifreeze compounds (5000) produced 95% hemolysis in red blood cells in the presence of glycerol. The low molecular weight glycopeptide compounds (3000) cause the same amount of hemolysis at a concentration of 4.0% w/v). Both the glycopeptide and peptide antifreezes cause ice growth along the c’axis, the nonpreferred axis of growth. The resulting spicular ice growth most likely produces shearing forces leading to disruption of the red blood cell membrane which would explain the absence of cryoprotection in the presence of glycerol and polyvinylpyrrolidone. 12. Cold Hardiness and Adaptive Potential of an Anlarctic Insect. J. G. BAUST (Department of
Biology, University Texas 77004).
of Houston,
Houston,
Befgica antarctica, a wingless fly and the only freeliving holometabolous insect of the Antarctic continent, is uniquely adapted for survival under perennial “winter conditions.” Larvae are freezing tolerant throughout the year and elaborate a complex of cryoprotectants including glycerol, erythritol, glucose, sucrose, and trehalose. Maintenance on artificial diets indicates that “antifreeze” profiles have foodsource and temperature-dependent components. Direct utilization of dietary cryoprotectants is suggested. The terrestrial microhabitat of the mid-Antarctic Peninsula provides a novel thermostable environment. The annual range of habitat temperatures did not exceed -7 to 11°C during the 2-year study. Accordingly this species lacks adaptive plasticity common to arctic insects. The following indicators of chill tolerance did not vary seasonally or during acclimation attempts. Supercooling points remained constant at -5.7 5 O.S”C. Lower lethal temperatures were also constant at -10°C (LT 50), and prolonged exposure to 310°C resulted in impaired carbohydrate metabolism. The thermostability of the environment of this species has resulted in the evolution of an obligate insect homeotherm.
13. Cryopreser\*afion
of
Sc,histosoma
Mansoni
E. R. JAMES (London School of Hygiene and Tropical Medicine, Winches Farm Field Station, 395 Hatfield Road, St. Albans, Hertfordshire AL4 OXQ, United Kingdom).
Schistosomula.
Schistosomula obtained by in vitro transformation of cercariae have been cryopreserved successfully only by using a two-step cooling schedule. During the first cooling step nucleation must occur at or close to the
MEETING
freezing point of the suspending medium, approximately - 10.5”C, since supercooling by even 1.5”C before nucleation leads to an eight-fold drop in survival. Below the freezing point survival declines progressively as the temperature is lowered, being lost completely by -40°C. However, samples cooled rapidly to - 196°C from intermediate temperatures below about -20°C yield some viable organisms on thawing. The best intermediate temperature depends chiefly on the cooling rate of the first step and on the type and concentration of cryoprotectant; the faster the cooling rate the lower the intermediate temperature required for peak survival. The optimum combination appears to be 1°C min-’ to -30°C using 4.56 M methanol. With 2.3 M ethanediol, the peak is lower and occurs at -20°C. The second cooling step is required to be as fast as possible, approximately 10,OOO”Cmin-’ in these studies. Rapid warming, at approximately 10,OOO”C min-‘, coupled with concomitant dilution to remove the cryoprotectant is necessary to achieve any survival. These results suggest that avoidance of intracellular ice formation during the first cooling step is essential; that the number of undamaged schistosomula is depleted during the slow cooling step probably as a result of osmotic stress and/or salt and cryoprotectant toxicity, as well as intracellular ice formation; that intracellular ice is induced in all organisms during the second cooling step: and that rapid warming is necessary to minimise the damage due to this intracellular ice. The post-in vitro transformation age is important also, peak survival occurring with organisms 60 to 90 min old: this phenomenon probably results from the complex physiological and morphological changes which accompany the transition from a free-living (cercaria) to a parasitic (schistosomulum) existence. With all the conditions optimal, approximately 30% of cryopreserved schistosomula show normal motility and following injection into mice 6.1% develop to adult worms (compared to approximately 30% for unfrozen organisms), indicating approximately 20% are infective. SESSION 14. Supercooling organic
2. CELL
BIOLOGY
und Supersafuration Salt Solutions.
ofAqueous
In-
A. P. MACKENZIE (Center for Bioengineering RF-52, University of Washington, Seattle, Washington 98195).
Previous studies (MacKenzie, A. P., Cryobiology 14, 705-706, 1977) showed that dilute aqueous NaCl solutions readily supercooled lo- 15°C and that eutectic crystallization was not usually seen above -35°C. Since totally solidified aqueous NaCl underwent an equilibrium eutectic melting at -2l”C, it was clear that NaCl supersaturated in practice in the presence of ice. Supercoolings of IO to 15°C suggested heterogeneous nucleation of ice. Whether or not the nucleation of the NaCI was heterogeneous or homogeneous was not determined. We now report experiments in which the