Freezing intracellular

If freezing of tissues occurs slowly, ice forms in extracellular areas as water flows out of the cell by exosmosis. As a result, the cell dehydrates and does not freeze intracellularly. However, if the cell is cooled rapidly, it cannot lose water fast enough to maintain equilibrium with its environment, and it therefore becomes increasingly supercooled and eventually freezes intracellularly (27). Mazur (27,28) suggested that injury from intracellular ice and its subsequent growth by recrystallization is a direct... 

Figure 2. Computed kinetics of water loss from mouse ova cooled at 1 °C to 32 °C/min in 1M DMSO. The curve labeled EQ shows the water content that ova have to maintain to remain in equilibrium with extracellular ice. If ova or embryos contain more than equilibrium amounts of water when they cool to below -30 °C, they will undergo intracellular freezing. Usually such freezing is lethal, but if the quantity of ice is small, some internally frozen cells can be rescued by rapid warming. (From Mazur, 1990.)...

Figure 3. Intracellular freezing of 8-cell mouse embryos cooled at 20 °C/min in 2 M DMSO. The black "flashing" occurring in cells at -31 °C to -46 °C is characteristic of intracellular ice formation, and is caused by the scattering of light by many small highly branched ice crystals. (Modified from Rail et al., 1983.)...

Figure 4. Percentage survival (dashed lines) vs. percentage cells undergoing intracellular freezing (solid lines) in three mammalian cells frozen at various rates to -20 °C (HeLa) or to -78 °C to -196 °C (ova and red blood cells (RBC)). (Modified from Leibo, 1977. Sources of data for individual curves are given there.)...

Mazur, P. (1963). Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J. Gen. Physiol. 47, 347-369. 

Mazur, P. (1977a). The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiol. 14,251-272. 

Continued intracellular freezing Recrystallization Hypoosmolality Indirect effects Tissue anoxia Thrombosis Edema Inflammation... 

With the suggestion that the last common genetic ancestor is a hyperthermophile, the role of temperature on the origins of life is important. The lower temperature limit in water is limited by the phase transition from liquid to ice. This is a problem because the density of ice is lower than that of water and the increase in volume on freezing will cause the cell structure to become disrupted in the same way that pipes burst in the winter. The lower limit for bacterial growth reported so far is -20°C, which is the temperature at which intracellular ice is formed. Adaptation to the cold requires a considerable salt content to depress the melting point of water the Don Juan Pond in Antarctica, which has a saturated CaCE solution, preserves the liquid phase at temperatures as low as —53°C. 

Figure 8.7 Overview of the manufacture of Betaferon, a recombinant human IFN-(3 produced in E. coli. The product differs from native human IFN-(3 in that it is unglycosylated and cysteine residue 17 had been replaced by a serine residue. E. coli fermentation is achieved using minimal salts/glucose media and product accumulates intracellularly in inclusion body (IB) form. During downstream processing, the lbs are solubilized in butanol, with subseguent removal of this denaturant to facilitate product refolding. After two consecutive gel-filtration steps, excipients are added, the product is filled into glass vials and freeze-dried. It exhibits a shelf life of 18 months when stored at 2-8 °C...

Fig. 1.20. Percentage of rat hepatocytes which show intracellular ice as a function of freezing rate in the range of -1 °C to -21 °C. The values in [ ] are the numbers of hepatocytes participating in each test (Fig. 8 from [ 1.28]).

Cryoinjury to the specimen is caused directly by extra- or intracellular ice crystal formation as well as by ice-induced solution effects during cryopreservation. Ice crystals seriously deform cell components. Another disadvantage of the formation of ice crystals near the specimen surface is slowing the cooling rate in areas below the surface because their thermal conductivity is about half that of solid water in a noncrystalline state. Furthermore, ice crystal formation is accompanied by the generation of latent heat, which also slows down the freezing rate. 

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