Vitrification of Carotid Artery Segments: An Integrated Study of Thermophysical Events and Functional Recovery Toward Scale-Up for Clinical Applications

Abstract

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 °C/min) or a low (2-3 °C/min) cooling rate, between -40°C and -100°C, and a high rewarming rate between -100°C and -40°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 (350%) 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.

Figure 1

Schematic diagram of the sequential incubation steps, adapted from a previously reported technique for adding and removing the vitrification cocktail, VS55 [24]. For artery rings, the loading and unloading of the cryoprotectant mixture was accomplished by immersing of the rings in the pre-cooled solutions (passive diffusion) for 15mins at each concentration step. For vessel segments, the process was facilitated by gravity perfusion of the solutions through the lumen of the artery segment [25]. Similarly, after vitrification and rewarming, the tissue samples were returned to physiological medium by eluting the cryoprotectants sequentially, using stepped changes. The cryoprotectant solution used at each step in the dilution was supplemented with 300mM mannitol as an osmotic buffer, to prevent excess cell swelling.Schematic diagram of the sequential incubation steps, adapted from a previously reported technique for adding and removing the vitrification cocktail, VS55.

Figure 2

Representative cryomacroscopy images of a blood vessel segment (its centerline is represented by a dash line) immersed in VS55, subject to H1=6.3°C/min, H2=4.4°C/min, H3=18.2°C/min, and H4=137.1°C/min, and minimum temperature of −137.2°C; top-left: the specimen surrounded by CPA at the beginning of cooling (−20°C); top-right: the specimen is surrounded by crystallized material, while the remaining of the domain is vitrified (−131.1°C); bottom-left: fracture formation in the glassy region of the VS55, at the top-center portion of the image; bottom-right: complete devitrification during rewarming (−89.9°C).

Figure 3

Representative cryomacroscopy images of vitrification of a blood vessel segment. at a minimum temperature of −132.2°C (top, the arrows point to the ends of the specimen and the dot line represents its centerline), and rings at a temperature of −131.4°C (bottom, selected rings are circled). In either image, the visibility of the polar grid underneath the CPA indicates that the VS55 vitrified, in contrast to the bottom-right image of Fig. 2.

Figure 4

Typical thermal protocols during the vitrification process on the cryomacroscope setup. Details on the experimental set-up used to achieve these thermal protocols are summarized in Table 1.

Figure 5

Functional recovery of artery segments and small artery rings after either the high or low cooling rate protocol (see Table 1). Function was assessed as either contractile or relaxation responses to pharmacological agonists (norepinephrine [N], phenylepinephrine [P]) or antagonists (calcium ionophore [Ca-I], sodium nitroprusside [SNP]), respectively, and normalized to the responses of control, untreated, tissue samples; controls were obtained from the same artery in each experiment. Data is expressed as the mean ± SEM.

Figure 6

Viability of artery rings and segments after vitrification, using either the high cooling rate (top), or low cooling rate protocol (bottom). Viability was determined using the alamarBlue metabolic indicator. Data was normalized with respect to fresh control tissue samples, obtained from the same artery as the vitrified samples. Data is expressed as the mean ± SEM.