J. Frim

Survival of unprotected, mammalian plateau-phase cells following freezing in liquid nitrogen.

Unprotected, mammalian cells in plateau phase are at least a factor of four times more sensitive to freeze-thaw damage than exponential-phase cells. The former suffer about 15-20% more sublethal damage after one freeze-thaw cycle than the latter and repair this damage more slowly. Exposure of plateau-phase cells to freeze-thaw damage lengthens the time required to traverse the cell cycle in the exposed generation. These cells may more closely represent the state in tissues than exponential-phase populations.

The effect of membrane lipid perturbers on survival of mammalian cells to cold.

Butylated hydroxytoluene (BHT), an antioxidant and common food additive, is an organic soluble molecule which modifies the properties of lipid bilayers and biological membranes. Adamantane and its derivatives, although structurally quite different, have similar effects on membranes. When Chinese hamster lung cells (V79) were pretreated with 0.1 mm BHT before exposure to +5 °C for up to 12 days in suspension culture or attached to plastic, significant protection, as assessed by colony survival, was observed compared to controls. No protection was observed when the cells were exposed to +20 °C. Use of adamantane or 2-adamantanone in serum-free medium in suspension culture at +5 °C showed protection of cells as good as, or better than, that provided by the addition of serum to control cells. Experiments with synchronized cells indicated that BHT protected cells in all phases of the cell cycle against the effects of exposure to +5 °C. However, the protection was greatest in G1 and early S phase. Exposure of cells containing BHT to +20 °C resulted in no preferential protection in any phase of the cell cycle.

Growth kinetics of cells following freezing in liquid nitrogen

Abstract The growth kinetics of cells frozen to −196 °C were monitored after thawing by various techniques. Progression through the cell cycle in the exposed generation was observed by monitoring cell growth either via multiplicity counts or by electronic cell counts of trypsinized suspensions. Subsequent generations were followed by time-lapse microcinematography. The division delay in the exposed generation of exponential-phase cells was dependent on cell age at the time of freezing and varied from 4 to 8 hr. The time of the first generation was still prolonged significantly but subsequent generations revealed cell cycle times that are comparable to unfrozen cells. In the case of plateau-phase cells, mitosis was delayed 7 hr in the exposed generation. This is 50% longer than the delay seen for pre-DNA synthetic g 1 cells in exponentially growing cultures. A rather important observation in this study was that frozen-thawed cells which divide once will probably continue dividing whereas eventual nonsurvivors are not likely to divide at all. The latter, however, remain active for more than 35 hr as observed microscopically, hence possibly indicating residual metabolic activity.

Factors affecting the repair of sublethal freeze-thaw damage in mammalian cells: I. Suboptimal temperature and hypoxia

Abstract The repair of sublethal freeze-thaw damage has been demonstrated by exposing cells to two freeze-thaw cycles separated by various times at 37 °C. The influence of suboptimal temperatures and hypoxia on this repair process was investigated. Repair was found to be temperature sensitive, decreasing as the temperature was lowered from 37 °C, and was virtually nonexistent at 5 °C. Lowering the oxygen concentration in the medium to 6.6 μ M or to 0.66 μ M had no significant effect on repair. The repair system then appears to involve an enzymatic process which is not greatly dependent on the oxygen concentration in the range of 277 (aerobic) to 0.66 μ M O 2 in the medium.

Factors affecting the repair of sublethal freeze-thaw damage in mammalian cells: II. The effect of ouabain☆

Abstract Mammalian cells were able to repair sublethal damage sustained during exposure to freeze-thaw conditions if they were incubated at 37 °C during the repair period. Repair was also observed when the cells were incubated at 37 °C in medium containing 10 −4 m ouabain but this was not the case with 10 −3 m ouabain. Cells exposed to either 10 −3 or 10 −4 m ouabain before freezing and thawing showed reduced survival indicating the requirement for the prior operation of the (Na + − K + ) — ATPase system to avoid additional lethal damage.

Freeze-thaw survival of the free-living nematode Caenorhabditis briggsae

Abstract Approximately 75% or more of the L2 and L3 juvenile stages of the free-living nematode Caenorhabditis briggsae survived freezing and thawing without loss of fertility. Optimum survival depended upon a combination of conditions: (1) pretreatment with 5% DMSO at 0 °C for 10 min, (2) 0.2 °C per minute cooling rate from 0 to −100 °C prior to immersion into liquid nitrogen, and (3) a 27.6 °C per minute warming rate from −196 °C to −10 °C. Storage at −196 °C for more than 100 days was without effect on viability or fertility. Some of the L4 (about 50%) and adult (about 3%) stages survive the routine freeze-thaw treatment. However, there was no recovery of either embryonic stages or embryonated eggs from −196 °C under these standard conditions. Either very fast cooling (about 545 °C/min) or fast warming (about 858 °C/min) rates diminished survival of the L2 and L3 stages drastically. Scanning electron microscopy revealed that freeze-thaw survivors with aberrant swimming behavior had cuticular defects. In juvenile forms, the altered swimming motion was lost after a molt whereas as abnormal adults grew, sinusoidal movement resumed. In the L4 and adult forms the cuticular abnormalities lowered viability and fertility. It is concluded that survival of nematodes from a freeze-thaw cycle is contingent upon establishing specific cryobiological conditions by varying aspects of the procedure that gave high recoveries of L2 and L3 stages.

Controlled variable-rate freeze-thaw apparatus.

Abstract The apparatus described above is an economic approach to controlled-rate cooling and warming for small sample sizes. It is inexpensive to build, very economical to run, yet flexible enough to allow a wide variety of cooling profiles to be generated. It was designed primarily to assist the user in achieving linear cooling at slow rates and it does this reproducibly and precisely.