Effect of Ice Nucleation and Cryoprotectants during High Subzero-Preservation in Endothelialized Microchannels

Abstract

Cryopreservation is of significance in areas including tissue engineering, regenerative medicine, and organ transplantation. We investigated endothelial cell attachment and membrane integrity in a microvasculature model at high subzero temperatures in the presence of extracellular ice. The results show that in the presence of heterogeneous extracellular ice formation induced by ice nucleating bacteria, endothelial cells showed improved attachment at temperature minimums of −6 °C. However, as temperatures decreased below −6 °C, endothelial cells required additional cryoprotectants. The glucose analog, 3-O-methyl-D-glucose (3-OMG), rescued cell attachment optimally at 100 mM (cells/lane was 34, as compared to 36 for controls), while 2% and 5% polyethylene glycol (PEG) were equally effective at −10 °C (88% and 86.4% intact membranes). Finally, endothelialized microchannels were stored for 72 h at −10 °C in a preservation solution consisting of the University of Wisconsin (UW) solution, Snomax, 3-OMG, PEG, glycerol, and trehalose, whereby cell attachment was not significantly different from unfrozen controls, although membrane integrity was compromised. These findings enrich our knowledge about the direct impact of extracellular ice on endothelial cells. Specifically, we show that, by controlling the ice nucleation temperature and uniformity, we can preserve cell attachment and membrane integrity. Further, we demonstrate the strength of leveraging endothelialized microchannels to fuel discoveries in cryopreservation of thick tissues and solid organs.

The atomistic details of the ice recrystallisation inhibition activity of PVA

“…IR damage can be mitigated by the use of cryoprotectants, but their usage suffers from extensive work protocols and potential cytotoxicity 5 , 8 – 10 . Consequently, a substantial body of work 5 , 6 , 10 – 17 has been devoted to finding alternative cryoprotectants that are biocompatible, relatively straightforward to produce and which display exceptional ice recrystallisation inhibition (IRI) activity at comparatively low concentrations.…”

Zebrafish as a New Tool in Heart Preservation Research

“…However, the supercooled state is thermodynamically unstable, meaning every degree decrease in temperature increases the risk of accidental ice formation, thereby limiting the depth of metabolic stasis that can be achieved. To further prolong cold ischemia time beyond supercooling preservation, others are suggesting an approach dubbed “partial freezing” that mimics freeze-tolerant wood frogs in nature [ 47 ] and aims to store whole organs at ~−20 °C for weeks [ 8 , 12 , 48 ]. However, partial freezing stores organs in the presence of ice, which can be exceptionally damaging if not properly controlled.…”

“…Extending the preservation duration of hearts would eliminate circumstantial factors, such as the timing of donor death and recipient/donor location, that contribute to a high rate of discarded organs, reduce the cost of transplantation, convert from emergency to planned surgeries, improve recipient outcomes by matching donors across greater distances, and enable tolerance induction protocols. However, after decades of successful heart transplantations, only modest increases in cardiac preservation duration have been achieved clinically, despite bold new solutions proposed in the literature, including several sub-zero approaches [ 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. Additionally, reviving “marginal” organs, especially from non-heart-beating donors, is another promising method [ 15 ].…”

New Approaches to Cryopreservation of Cells, Tissues, and Organs

“…Importantly, research has shown that simple augmentation strategies such as increasing endogenous levels of cryoprotectants (via injection) prior to freezing have the capacity to improve survival and extend the time in a frozen state in a certain context from 2 weeks to 49 days [113]. A translation of nature’s cryostasis strategies to human systems may be achieved through low-molecular-weight CPAs, in particular the aforementioned nonmetabolizable glucose derivative 3-OMG and oligosaccharides, of which trehalose has received notable attention in recent years [54, 115, 116]. …”

“…It therefore has been hypothesized that through the application of biocompatible ice nucleators, controlled, slow propagation of freezing at relatively high subzero temperatures can be achieved and thereby cryodamage be minimized. Preliminary support for this approach in a simple blood vessel model has recently been provided [116]. …”