The limited preservation duration of organs has contributed to the shortage of organs for transplantation. Recently, a tripling of the storage duration was achieved with supercooling, which relies on temperatures between −4 and −6 °C. However, to achieve deeper metabolic stasis, lower temperatures are required. Inspired by freeze-tolerant animals, we entered high-subzero temperatures (−10 to −15 °C) using ice nucleators to control ice and cryoprotective agents (CPAs) to maintain an unfrozen liquid fraction. We present this approach, termed partial freezing, by testing gradual (un)loading and different CPAs, holding temperatures, and storage durations. Results indicate that propylene glycol outperforms glycerol and injury is largely influenced by storage temperatures. Subsequently, we demonstrate that machine perfusion enhancements improve the recovery of livers after freezing. Ultimately, livers that were partially frozen for 5-fold longer showed favorable outcomes as compared to viable controls, although frozen livers had lower cumulative bile and higher liver enzymes.
- Award ID(s):
- 1941543
- NSF-PAR ID:
- 10471549
- Publisher / Repository:
- NSF
- Date Published:
- Journal Name:
- Frontiers in Physics
- Volume:
- 10
- ISSN:
- 2296-424X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Dryophytes chrysoscelis (formerlyHyla chrysoscelis , Cope’s gray treefrog) is a freeze‐tolerant anuran that accumulates glycerol and urea during cold acclimation and freezing. It is hypothesized that glycerol and urea function as cryoprotectants by minimizing osmotically induced cell damage during freezing and thawing, thereby improving the postfreeze viability of red blood cells (RBCs) when frozen in medium containing those solutes. To test this, erythrocytes were obtained from warm (22°C) and cold‐acclimated (4°C) frogs and suspended in 280 mOsM phosphate‐buffered saline (PBS). RBCs were frozen in 280 mOsM, isosmotic/isotonic, PBS, or in PBS made hyperosmotic by addition of 150 mM solutes. Postfreeze viability was determined with a hemolysis assay. Postfreeze viability of cells from warm‐acclimated frogs improved from 18.9 ± 1.3% in PBS to 47.4 ± 5.2% in PBS with urea (p < 0.01). The addition of other solutes (glycerol, glucose, NaCl, or sorbitol) had no effect. RBCs from cold‐acclimated frogs had 45.8 ± 3.4% viability when frozen in 280 mOsM PBS, and this improved to 71.6 ± 8.9% or 71.9 ± 1.6%, respectively, when frozen with glycerol (p < 0.01) or urea (p < 0.001). The viability of RBCs from cold‐acclimated frogs was not different between unfrozen cells 86.7–88.4%) and those frozen with glycerol (71.6 ± 8.9%,p > 0.05) or with urea (71.9 ± 1.6%,p > 0.05). These data suggest that (a) cold acclimation induces cellular changes in RBCs that result in improved postfreeze viability, and (b) glycerol and urea are part of a complex cryoprotectant system inD. chrysoscelis. -
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Additives that help cells survive the stresses of freezing and thawing are known as cryoprotective agents (CPAs). Two different types of CPAs have been identified: penetrating and non-penetrating. Common penetrating CPAs include dimethylsulfoxide (DMSO) and glycerol. The location of a CPA (intracelluar or extracellular) is important for understanding the molecular mechanisms of action for the agent. Low-temperature Raman spectroscopy is a label-free method of detecting the location of CPAs at low temperature with high spatial resolution and chemical specificity. To this end, cells cryopreserved in DMSO using a variety of cooling rates and DMSO concentrations and imaged using Raman spectroscopy were analyzed using automated image analysis to determine the partitioning ratio (concentration of DMSO outside/concentration of DMSO inside the cell). The partitioning ratio was roughly 1 for Jurkat cells frozen at 1°C/min in varying concentrations of DMSO with the exception of 1% DMSO which had a partitioning ratio of 0.2. The partitioning ratio increased from 1 to 1.3 as the cooling rate increased from 1°C to 5°C/min. Different cell types, specifically sensory neurons cells and human induced pluripotent stem cells, exhibited differences in partitioning ratio when frozen in 10% DMSO and 1°C/min suggesting that differences in freezing response may result from differences in solute partitioning. The presence of intracellular ice changed the distribution of DMSO inside the cell and also the partitioning ratio.
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Abstract In transplantation, livers are transported to recipients using static cold storage (SCS), whereby livers are exposed to cold ischemic injury that contribute to post-transplant risk factors. We hypothesized that flushing organs during procurement with cold preservation solutions could influence the number of donor blood cells retained in the allograft thereby exacerbating cold ischemic injury. We present the results of rat livers that underwent 24 h SCS after being flushed with a cold University of Wisconsin (UW) solution versus room temperature (RT) lactated ringers (LR) solution. These results were compared to livers that were not flushed prior to SCS and thoroughly flushed livers without SCS. We used viability and injury metrics collected during normothermic machine perfusion (NMP) and the number of retained peripheral cells (RPCs) measured by histology to compare outcomes. Compared to the cold UW flush group, livers flushed with RT LR had lower resistance, lactate, AST, and ALT at 6 h of NMP. The number of RPCs also had significant positive correlations with resistance, lactate, and potassium levels and a negative correlation with energy charge. In conclusion, livers exposed to cold UW flush prior to SCS appear to perform worse during NMP, compared to RT LR flush.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fphy.2022.1033613
