- Email: [email protected]
Optimizing conditions for cryopreservation of an insect cell line
ABSTRACTS,
2lst ANNUAL
ington. Seattle, Washington 98105, and Department of Biology, University of Notre Dame. Notre Dame, Indiana 46556). Several species of insects produce thermal hysteresis proteins (THPs) in the winter which function as antifreezes. Photoperiod has been shown to be an important trigger for antifreeze protein production in the beetle. Lkndroides cctnadensis. Also, the onset of lower temperature is capable of modifying the photoperiodic response and increasing levels of THPs. This study considers a possible role for thermoperiods (i.e.. the duration of thermophase (T) and cryophase (C) during a 24-hr period) in the regulation of THP5 in D. Cuwden.sis. To see if thermoperiod influences induction of THP production larvae were collected in earl! fall from a natural population and maintained under constant light (LL) conditions at 20°C (show’n previously to prevent elevation of THPs). Animals maintained in LL for 3 weeks while being exposed to a long-day thermoperiod (T = I6 hr at 2S”Ci c‘ = 8 hr at 17°C) possessed low THP levels. However. individuals placed in short-day thermoperiodic conditions (T = 8 hr at 25°C i C = I6 hr at 17°C) under LL significantly (P < .Ol) elevated antifreeze protein levels. To examine the role of thermoperiod in the prevention of THP production larvae were also maintained under constant darkness (DD) at 20°C (These conditions are known to elevate THP levels). Exposure of D. (‘(,,?(Idrmis to a long-day thermoperiod and DD prevented the increase in THP levels while those held on a shortday thermoperiod showed a significant (P < .Ol) elevation in the antifreezes. These results indicate that are capable of distinguishing larvae of D. cancrdrnsis long- from short-day thermoperiods and initiating antifreeze protein production under the appropriate conditions. Further experiments in which the cryophase was allowed to reach 13°C (near the critical temperature for triggering THP production independently of the photoregime) were then conducted to study the influence of temperature on this thermoperiodic response. Significant differences between long- and short-day thermoperiodic responses persisted, while the overall levels of THPs increased for all groups. Finally. preliminary experiments using conflicting photoperiodic and thermoperiodic regimes were conducted. Intermediate responses in elevated THP levels were observed under the conflicting experimental conditions employed suggesting perhaps that each factor plays an important role in the regulation of antifreeze protein production. 16. Optimizing
Conditions
,for C~~op/.~.se~~,rrrion
of trn
Cell Lir7e. A. E. HEACOX AND R. A. LEOPOLD (Department of Zoology. North Dakota State University, Fargo. North Dakota 58102, and U.S. Department of Agriculture. Agricultural Research Service, Metabolism and 1~7sc~t
687
MEETING Radiation Dakota).
Research
Laboratory.
Fargo, North
A cell line (UM-BGE-2) derived from embryos of the cockroach. BlatreJltr gern2rrnic.u. was frozen to - 196°C under a variety of conditions and cell viability was assayed after warming. It was found that cell viability was affected by the cooling rate, the warming rate, the controlled cooling end point temperature. and the type and concentration of cryoprotectant. The best survival for cells suspended in Grace’s tissue culture medium containing I IV MeXl was oblained when cells were cooled at I”C/min to at leabt -90°C before being placed in liquid nitrogen and warmed at more than YOO”Cimin. Cultures initiated from these frozen cells produce typical growth curves and appear normal after several passages. SESSION
I-SYMPOSIUM:
THE ISOLATED 17.
PHYSIOLOGY
PERFUSED
OF
ORGAN
of’ tl7e Isoluted Prrfirsc~d Kidwy. L. (Department of Medicine. University of Colorado Health Science Center. Denver, Colorado 80261).
P/~~v;o/o,~~
CHAN
18.
P/7~sio/og~
Il.\
of‘thr Isolatrd Pcvfusc~d Dog Liver cmd Use in Orgcrn Pwsurvcrficm. L. LAMB01.r~.
(Laboratory for Experimental Surgery. UCL 5570, avenue Hippocrate, 55. 1200 Brussels, Belgium). Liver perfusion was first used as a method of organ preservation. perfusion in hypothermia with blood, cryoprecipitated plasma. and sometimes with hyperbaric oxygenation. Because the method was very complex and did not yield very good results. it was given up. Liver perfusion was then applied as a substitute for actual transplantation in order to evaluate more easily new methods of preservation as. for instance. some pharmacologic protective agents. For this purpose. various tests of liver function were needed: a few were based on specific liver function (bile production. BSP elimination) but most relied on basic cellular mechanisms. We have introduced the measurement of membrane potential which is related to membrane permeability and active ion transport. It has been used to show that isoproterenol increases the recovery of liver function after a temporary ischemia and has been applied to transplantation (L. Lambotte. .Sl~rgc~r~74, 509-518. 1973). Liver perfusion has then been used to obtain a better understanding of the mechanism of action of the intracellular hyperosmolar solution. It was shown that, after a 24.hr preservation period. the swelling was partially inhibited with some of these solutions. but the energy metabolism was not markedly modified since the mitochondria were able to sustain such a period of anoxia (L. Lambotte and S. Wojcik. Slrr~eq 83, 94-103. 1978; S. Pontegnie-
2lst ANNUAL
ington. Seattle, Washington 98105, and Department of Biology, University of Notre Dame. Notre Dame, Indiana 46556). Several species of insects produce thermal hysteresis proteins (THPs) in the winter which function as antifreezes. Photoperiod has been shown to be an important trigger for antifreeze protein production in the beetle. Lkndroides cctnadensis. Also, the onset of lower temperature is capable of modifying the photoperiodic response and increasing levels of THPs. This study considers a possible role for thermoperiods (i.e.. the duration of thermophase (T) and cryophase (C) during a 24-hr period) in the regulation of THP5 in D. Cuwden.sis. To see if thermoperiod influences induction of THP production larvae were collected in earl! fall from a natural population and maintained under constant light (LL) conditions at 20°C (show’n previously to prevent elevation of THPs). Animals maintained in LL for 3 weeks while being exposed to a long-day thermoperiod (T = I6 hr at 2S”Ci c‘ = 8 hr at 17°C) possessed low THP levels. However. individuals placed in short-day thermoperiodic conditions (T = 8 hr at 25°C i C = I6 hr at 17°C) under LL significantly (P < .Ol) elevated antifreeze protein levels. To examine the role of thermoperiod in the prevention of THP production larvae were also maintained under constant darkness (DD) at 20°C (These conditions are known to elevate THP levels). Exposure of D. (‘(,,?(Idrmis to a long-day thermoperiod and DD prevented the increase in THP levels while those held on a shortday thermoperiod showed a significant (P < .Ol) elevation in the antifreezes. These results indicate that are capable of distinguishing larvae of D. cancrdrnsis long- from short-day thermoperiods and initiating antifreeze protein production under the appropriate conditions. Further experiments in which the cryophase was allowed to reach 13°C (near the critical temperature for triggering THP production independently of the photoregime) were then conducted to study the influence of temperature on this thermoperiodic response. Significant differences between long- and short-day thermoperiodic responses persisted, while the overall levels of THPs increased for all groups. Finally. preliminary experiments using conflicting photoperiodic and thermoperiodic regimes were conducted. Intermediate responses in elevated THP levels were observed under the conflicting experimental conditions employed suggesting perhaps that each factor plays an important role in the regulation of antifreeze protein production. 16. Optimizing
Conditions
,for C~~op/.~.se~~,rrrion
of trn
Cell Lir7e. A. E. HEACOX AND R. A. LEOPOLD (Department of Zoology. North Dakota State University, Fargo. North Dakota 58102, and U.S. Department of Agriculture. Agricultural Research Service, Metabolism and 1~7sc~t
687
MEETING Radiation Dakota).
Research
Laboratory.
Fargo, North
A cell line (UM-BGE-2) derived from embryos of the cockroach. BlatreJltr gern2rrnic.u. was frozen to - 196°C under a variety of conditions and cell viability was assayed after warming. It was found that cell viability was affected by the cooling rate, the warming rate, the controlled cooling end point temperature. and the type and concentration of cryoprotectant. The best survival for cells suspended in Grace’s tissue culture medium containing I IV MeXl was oblained when cells were cooled at I”C/min to at leabt -90°C before being placed in liquid nitrogen and warmed at more than YOO”Cimin. Cultures initiated from these frozen cells produce typical growth curves and appear normal after several passages. SESSION
I-SYMPOSIUM:
THE ISOLATED 17.
PHYSIOLOGY
PERFUSED
OF
ORGAN
of’ tl7e Isoluted Prrfirsc~d Kidwy. L. (Department of Medicine. University of Colorado Health Science Center. Denver, Colorado 80261).
P/~~v;o/o,~~
CHAN
18.
P/7~sio/og~
Il.\
of‘thr Isolatrd Pcvfusc~d Dog Liver cmd Use in Orgcrn Pwsurvcrficm. L. LAMB01.r~.
(Laboratory for Experimental Surgery. UCL 5570, avenue Hippocrate, 55. 1200 Brussels, Belgium). Liver perfusion was first used as a method of organ preservation. perfusion in hypothermia with blood, cryoprecipitated plasma. and sometimes with hyperbaric oxygenation. Because the method was very complex and did not yield very good results. it was given up. Liver perfusion was then applied as a substitute for actual transplantation in order to evaluate more easily new methods of preservation as. for instance. some pharmacologic protective agents. For this purpose. various tests of liver function were needed: a few were based on specific liver function (bile production. BSP elimination) but most relied on basic cellular mechanisms. We have introduced the measurement of membrane potential which is related to membrane permeability and active ion transport. It has been used to show that isoproterenol increases the recovery of liver function after a temporary ischemia and has been applied to transplantation (L. Lambotte. .Sl~rgc~r~74, 509-518. 1973). Liver perfusion has then been used to obtain a better understanding of the mechanism of action of the intracellular hyperosmolar solution. It was shown that, after a 24.hr preservation period. the swelling was partially inhibited with some of these solutions. but the energy metabolism was not markedly modified since the mitochondria were able to sustain such a period of anoxia (L. Lambotte and S. Wojcik. Slrr~eq 83, 94-103. 1978; S. Pontegnie-