The mechanisms behind slow freezing injury to cells and the protective role of solutes like glycerol are still actively discussed in the scientific community. During a slow freezing process, cells find themselves confined within unfrozen channels formed between ice crystals. These ice crystals enlarge by extracting pure water from these channels. This process leads to a significant increase in the solute concentration within these channels, and consequently, a progressive reduction in the volume of liquid space available. This elevation in solute concentration causes cells to undergo osmotic shrinkage. Traditionally, cryobiologists have attributed slow freezing injury to either the elevated concentrations of solutes (electrolytes) within these channels or to the subsequent cell shrinkage. These theories often overshadowed the direct impact of the diminishing size of the channels themselves.
However, it’s crucial to understand that while channel composition and size are typically interconnected, they can be experimentally decoupled. This separation can be achieved by suspending cells in solutions containing both NaCl and cryoprotectants, maintaining a constant mole ratio between them, but varying the overall molality of NaCl around isotonic levels. Experiments conducted on human red blood cells frozen in such solutions, reaching temperatures that induce specific NaCl concentrations (ms) but varying unfrozen fractions (U), revealed significant insights. Survival rates at low U values were found to be strongly correlated with U, but surprisingly independent of ms, meaning The Solution Had Salt Concentration Compared To The Cell was not the primary factor in cell survival in these conditions. Conversely, at higher U values, cell survival became inversely dependent on both ms and U.
Interestingly, cell volume during freezing appeared to be unaffected by the NaCl tonicity of the solution. Despite this, the cells in different solutions exhibited varying volumes both before freezing commenced and after thawing was complete. Further investigation involved returning the thawed cells to isotonic solutions, aiming to restore their isotonic volume, or close to it. The results indicated that minimal change in survival was observed following exposure to low U values. However, after exposure to high U values, a notably increased sensitivity to ms emerged. This heightened sensitivity likely points to posthypertonic hemolysis, a phenomenon where cells rupture due to osmotic stress after being subjected to a hypertonic environment.
The study design inherently linked low U values to solutions with lower NaCl tonicities. As a result, cells frozen to low U values had larger initial volumes before freezing compared to cells frozen to higher U values. The implications of this interrelation are important to consider when interpreting these findings and understanding the complex interplay of factors determining cell survival during slow freezing.
