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Cryobiology

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Cryobiology 57 2008 1 8 Contents lists available at ScienceDirect Cryobiology journal homepage www elsevier com locate ycryo Cryopreservation of carotid artery segments via vitri cation subject to marginal thermal conditions Correlation of freezing visualization with functional recovery q S Baicu a M J Taylor a b Z Chen a Y Rabin b a b Cell and Tissue Systems Inc 2231 Technical Parkway Suite A N Charleston SC 29406 USA Department of Mechanical Engineering Carnegie Mellon University Pittsburgh PA 15213 USA a r t i c l e i n f o Article history Received 24 January 2008 Accepted 20 March 2008 Available online 28 March 2008 Keywords Vitri cation Functional recovery Blood vessels Cryomacroscopy Thermo physical events Carotid artery Ice visualization Cracking a b s t r a c t Cryopreservation is a well established technique for long term storage of viable cells and tissues However in recent years application of established cryobiological principles to the preservation of multicellular tissues and organs has demanded considerable attention to ways of circumventing the deleterious effects of ice and thermal stresses in bulky tissues As part of a multidisciplinary research program designed to study the interactions of thermo physical events with tissue preservation we report here on the implementation of a slow cooling 3 C min and slow warming 62 C min regimen towards scale up of vitreous preservation of large tissue samples Speci cally the correlation of thermo physical events during vitri cation of carotid artery segments with function recovery is reported using marginal thermal conditions for achieving vitri cation in bulky samples Moreover the outcome is compared with a similar study reported previously using a 3 fold higher rate of rewarming 186 13 C min Tissue vitri cation using an 8 4 M cryoprotectant cocktail solution VS55 was achieved in 1 ml samples by imposing a low 2 6 0 1 C min cooling rate between 40 C and 100 C and a low rewarming rate 62 4 C min between 100 C and 40 C Following cryoprotectant removal the artery segments were cut into 3 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 non speci c metabolic function of the vessel rings was determined using the REDOX indicator alamarBlue Contractile function normalized to untreated control samples in response to the agonists norepinephrine and phenylepinephrine was signi cantly better in the slowly rewarmed group of carotid segments 74 9 and 62 11 respectively than for the more rapidly warmed group 31 7 and 45 15 respectively However EC50 sensitivities were not signi cantly different between the groups Thermo physical events such as ice formation and fractures were monitored throughout the cooling and warming phases using cryomacroscopy with the aid of a purpose built borescope device This technique allowed a direct observation of the visual impact of ice formation on speci c zones along the blood vessel segment where in most cases no ice formation or fractures were observed in the vicinity of the artery segments However in speci c instances when some ice crystallization was observed to impact the artery segment the subsequent testing of function revealed a total loss of contractility The successful vitri cation of blood vessel segments using marginal conditions of slow cooling and rewarming provide essential information for the development of scale up protocols that is necessary when clinically relevant size samples need to be cryopreserved in an essentially ice free state This information can further be integrated into computer simulations of heat transfer and thermo mechanical stress where the slowest cooling rate anywhere in the simulated domain must exceed the critical values identi ed in the current study 2008 Published by Elsevier Inc Extrapolation of the successful techniques for cryopreservation of cell suspensions to multicellular tissues with a de ned architecq This study was supported by NIH NHLBI Grant No RO1HL06994401A1 02 03 04 Corresponding author Address Cell and Tissue Systems Inc 2231 Technical Parkway Suite A N Charleston SC 29406 USA Fax 1 843 722 6657 E mail address mtaylor organ recovery com M J Taylor 0011 2240 see front matter 2008 Published by Elsevier Inc doi 10 1016 j cryobiol 2008 03 002 ture has proven to be a challenging task due principally to the damaging effects of ice formation and development of thermomechanical stresses as reviewed recently by Taylor Rabin and co workers 3 14 In a series of studies on clinically relevant model systems such as a blood vessel and an articular cartilage a markedly improved cryopreservation outcome was demonstrated where the system is preserved in an essentially ice free state 2 S Baicu et al Cryobiology 57 2008 1 8 5 7 8 14 16 Having shown feasibility in small model systems for vitreous cryopreservation of complex tissues the research is now aimed at scale up experiments towards a clinically signi cant sample size This line of research relies upon a recently presented study designed to vitrify carotid artery segments using as slow as possible cooling rates to suppress ice formation 1 This previous study included rapid rewarming where the rewarming rate was not a studied parameter 1 The current study complements the recently published report by exploring the minimum rewarming rates to ensure vitri cation The current study does not merely address critical rewarming rates of the cryoprotective agent nor the functional recovery post cryopreservation as an end result test but the correlation between the two different effects of the process By means of visualization thermo physical events of crystallization and possibly fracturing are correlated with the quality of the recovered specimen Promoting vitri cation while preventing structural damage represent competing needs whereas there are both minimum cooling and rewarming rates to ensure vitri cation and maximum cooling and rewarming rate to avoid structural damage Developing the knowledge about the possible balance between these competing needs serves as a prelude for attempting vitreous cryopreservation of bulky samples In order to explore the reasonable boundaries of cryopreservation via vitri cation a prototype imaging device termed a cryomacroscope was recently developed 4 12 to observe possible crystallization events and fracture formation in


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