Evaluation of freezing effects on human microvascular-endothelial cells (HMEC)

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. In addition, new catheter based cryosurgical probes are being designed for cardiovascular applications where endothelial and smooth muscle cell freezing is involved but not well understood. 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 which can affect post-freeze viability. The hMECs were loaded on a cryomicroscope stage and freezing experiments at 5, 10, 15, 25, 100 and 130 degrees C/min were performed to experimentally evaluate dehydration (water transport) as well as intracellular ice formation (IIF) within this cell system. The dehydration kinetics at 5, 10 and 25 degrees C/min were found to be governed by a membrane permeability L(pg) and activation energy E(Lp) of 0.05 (microm/min.atm) and 14.8 (kcal/mole) respectively [R(2)=0.94]. These parameters were then tested for predictive ability against the experimentally measured behavior at 15 degrees C/min with a good agreement [R(2)=0.98]. Intracellular Ice Formation (IIF) was found to occur at lower temperatures than many cell types (i.e. TIIF 50% approximately -18 degrees C) and at cooling rates greater than or equal to 25 degrees C/min. At cooling rates above 50 degrees 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, omega(o) and kappa(o), which fit data from 50, 100 and 130 degrees C/min were found to be 6.8 x 10(-8) (m(2).s)(-1) and 8.3 x 10(-9) (K5) [R(2)=0.94] respectively. Preliminary results show that viability drops precipitously between -20 and -30 degrees C, however, further studies are warranted to address the role of cooling rate, end-temperature, hold time and thawing rate to establish the freeze sensitivity of this cell.