Abstract

There is mounting evidence that the endothelium may play an important role in traditional cryosurgical treatments by acting to locally foster thrombi in the microvasculature of various tissues after freezing. Therefore, this study was designed to investigate, at the cellular level in human microvascular endothelial cells (hMEC), the various biophysical changes that occur during freezing and compare them with post-freeze viability. The hMECs were loaded on a cryomicroscope stage and freezing experiments at 5, 10, 15, 25, 100 and 130°C/min were performed to experimentally evaluate dehydration (water transport) as well as intracellular ice formation (IIF) within this cell system. The dehydration kinetics were found to be governed by a membrane permeability Lpg and activation energy ELp of 0.05 (μm/min.atm) and 14.8 (kcal/mole) respectively [R2 = 0.94]. These parameters were then tested for predictive ability against the experimentally measured behavior at 15°C/min with a good agreement [R2 = 0.98]. Intracellular Ice Formation (IIF) was found to occur at lower temperatures than many cell types (i.e. TIIF 50% ∼ −18°C) and at cooling rates greater than or equal to 25°C/min. At cooling rates above 50°C/min, two types of IIF, cell darkening and twitching, were both observed and quantified and were assumed to be governed by Surface Catalyzed Nucleation (SCN). IIF parameters Ωo, and κo were found to be 6.8 × 10−8 (m2.s)−1 and 8.3 × 10−9 (K5) [R2 = 0.94] respectively. Viability results suggest an inverted U-shape curve between 1 and 50°C/min (with a maximum at 10°C/min). But viability appears to increase again at cooling rates > 50°C/min (i.e. it does not continue to drop) which suggests that the traditional two factor hypothesis may not completely describe viability in this system. Additional cellular destruction was found by lowering the end-temperature to −30°C or below. At this temperature the majority of the cell population was destroyed regardless of the cooling rate.

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