Vitrification of carotid artery segments: An integrated study of thermophysical events and functional recovery toward scale-up for clinical applications

In recent years, ice-free cryopreservation by vitrification has been demonstrated to provide superior preservation of tissues compared with conventional freezing methods. To date, this has been accomplished almost exclusively for small model systems, whereas cryopreservation of large tissue samples-of a clinically useful size-continues to be hampered by thermomechanical effects that compromise the structure and function of the tissue. Reduction of mechanical stress is an integral condition of successful cryopreservation of large specimens. The current study focuses on the impact of sample size on both the physical events, observed by cryomacroscopy, and on the outcome on tissue function. To this end, the current study sought to address the question of functional recovery of vitrified carotid artery segments, processed as either artery rings (3-4 mm long) or segments (25 mm long) as selected models; the latter model represents a significant increase in sample size for evaluating the effects of vitrification. Tissue vitrification using an 8.4 M cryoprotectant cocktail solution (VS55) was achieved in 1-ml samples by imposing either a high (50-70 degrees C/min) or a low (2-3 degrees C/min) cooling rate, between -40 degrees C and -100 degrees C, and a high rewarming rate between -100 degrees C and -40 degrees C. Following cryoprotectant removal, the artery segments were cut into 3 to 4-mm rings for function testing on a contractility apparatus by measuring isometric responses to four agonist and antagonists (norepinephrine, phenylepinephrine, calcium ionophore, and sodium nitroprusside). In addition, nonspecific metabolic function of the vessel rings was determined using the REDOX indicator alamarBlue. Contractile function in response to the agonists norepinephrine and phenylepinephrine was maintained at the same level (similar to 50%) for the segments as for the rings, when compared with noncryopreserved control samples. Relaxation in response to the antagonists calcium ionophore and sodium nitroprusside was maintained at between 75% and 100% of control levels, irrespective of cooling rate or sample size. No evidence of macroscopic crystallization or fractures was observed by cryomacroscopy at the above rates in any of the samples. In conclusion, this study verifies that the rate of cooling and warming can be reduced from our baseline vitrification technique such that the function of larger tissue samples is not significantly different from that of smaller blood vessel rings. This represents a step toward the goal of achieving vitreous cryopreservation of large tissue samples without the destructive effect of thermal stresses.