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1. Partial freezing of rat livers extends preservation time by 5-fold

   https://doi.org/10.1038/s41467-022-31490-2  

   Tessier, Shannon N. ; de Vries, Reinier J. ; Pendexter, Casie A. ; Cronin, Stephanie E. J. ; Ozer, Sinan ; Hafiz, Ehab O. A. ; Raigani, Siavash ; Oliveira-Costa, Joao Paulo ; Wilks, Benjamin T. ; Lopera Higuita, Manuela ; et al ( July 2022 , Nature Communications)

   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.

    

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2. Postfreeze viability of erythrocytes from Dryophytes chrysoscelis

   https://doi.org/10.1002/jez.2262  

   Geiss, Loren ; do Amaral, M. Clara F. ; Frisbie, James ; Goldstein, David L. ; Krane, Carissa M. ( April 2019 , Journal of Experimental Zoology Part A: Ecological and Integrative Physiology)

   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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3. Cryopreserved red blood cells maintain allosteric control of oxygen binding when utilizing trehalose as a cryoprotectant

   https://doi.org/10.1016/j.cryobiol.2023.104793  

   Elder, Charles A. ; Smith, Jensen S. ; Almosawi, Mustafa ; Mills, Ethan ; Janis, Brett R. ; Kopechek, Jonathan A. ; Wolkers, Willem F. ; Menze, Michael A. ( March 2024 , Cryobiology)

   One of the most common life-saving medical procedures is a red blood cell (RBC) transfusion. Unfortunately, RBCs for transfusion have a limited shelf life after donation due to detrimental storage effects on their morphological and biochemical properties. Inspired by nature, a biomimetics approach was developed to preserve RBCs for long-term storage using compounds found in animals with a natural propensity to survive in a frozen or desiccated state for decades. Trehalose was employed as a cryoprotective agent and added to the extracellular freezing solution of porcine RBCs. Slow cooling (-1 C min-1) resulted in almost complete hemolysis (1 ± 1 % RBC recovery), and rapid cooling rates had to be used to achieve satisfactory cryopreservation outcomes. After rapid cooling, the highest percentage of RBC recovery was obtained by plunging in liquid nitrogen and thawing at 55 C, using a cryopreservation solution containing 300 mM trehalose. Under these conditions, 88 ± 8 % of processed RBCs were recovered and retained hemoglobin (14 ± 2 % hemolysis). Hemoglobin’s oxygen-binding properties of cryopreserved RBCs were not significantly different to unfrozen controls and was allosterically regulated by 2,3-bisphosphoglycerate. These data indicate the feasibility of using trehalose instead of glycerol as a cryoprotective compound for RBCs. In contrast to glycerol, trehalose-preserved RBCs can potentially be transfused without time-consuming washing steps, which significantly facilitates the usage of cryopreserved transfusible units in trauma situations when time is of the essence. 

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4. Comparing two extracellular additives to facilitate extended storage of red blood cells in a supercooled state

   https://doi.org/10.3389/fphys.2023.1165330  

   William, Nishaka ; Isiksacan, Ziya ; Mykhailova, Olga ; Olafson, Carly ; Yarmush, Martin L. ; Usta, O. Berk ; Acker, Jason P. ( June 2023 , Frontiers in Physiology)

   Background:Adenosine triphosphate (ATP) levels guide many aspects of the red blood cell (RBC) hypothermic storage lesions. As a result, efforts to improve the quality of hypothermic-stored red cell concentrates (RCCs) have largely centered around designing storage solutions to promote ATP retention. Considering reduced temperatures alone would diminish metabolism, and thereby enhance ATP retention, we evaluated: (a) whether the quality of stored blood is improved at −4°C relative to conventional 4°C storage, and (b) whether the addition of trehalose and PEG400 can enhance these improvements.

   Study Design and Methods:Ten CPD/SAGM leukoreduced RCCs were pooled, split, and resuspended in a next-generation storage solution (i.e., PAG3M) supplemented with 0–165 mM of trehalose or 0–165 mM of PEG400. In a separate subset of samples, mannitol was removed at equimolar concentrations to achieve a fixed osmolarity between the additive and non-additive groups. All samples were stored at both 4°C and −4°C under a layer of paraffin oil to prevent ice formation.

   Results:PEG400 reduced hemolysis and increased deformability in −4°C-stored samples when used at a concentration of 110 mM. Reduced temperatures did indeed enhance ATP retention; however, in the absence of an additive, the characteristic storage-dependent decline in deformability and increase in hemolysis was exacerbated. The addition of trehalose enhanced this decline in deformability and hemolysis at −4°C; although, this was marginally alleviated by the osmolarity-adjustments. In contrast, outcomes with PEG400 were worsened by these osmolarity adjustments, but at no concentration, in the absence of these adjustments, was damage greater than the control.

   Discussion:Supercooled temperatures can allow for improved ATP retention; however, this does not translate into improved storage success. Additional work is necessary to further elucidate the mechanism of injury that progresses at these temperatures such that storage solutions can be designed which allow RBCs to benefit from this diminished rate of metabolic deterioration. The present study suggests that PEG400 could be an ideal component in these solutions.

    

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5. Neurovascular injury associated non-apoptotic endothelial caspase-9 and astroglial caspase-9 mediate inflammation and contrast sensitivity decline

   https://doi.org/10.1038/s41419-022-05387-3  

   Colón Ortiz, Crystal ; Neal, Albertine M. ; Avrutsky, Maria I. ; Choi, Monica ; Smart, Jade ; Lawson, Jacqueline ; Troy, Carol M. ( November 2022 , Cell Death & Disease)

   Retinal neurovascular injuries are a leading cause of vision loss in young adults presenting unmet therapeutic needs. Neurovascular injuries damage homeostatic communication between endothelial, pericyte, glial, and neuronal cells through signaling pathways that remain to be established. To understand the mechanisms that contribute to neuronal death, we use a mouse model of retinal vein occlusion (RVO). Using this model, we previously discovered that after vascular damage, there was non-apoptotic activation of endothelial caspase-9 (EC Casp9); knock-out of EC Casp9 led to a decrease in retinal edema, capillary ischemia, and neuronal death. In this study, we aimed to explore the role of EC Casp9 in vision loss and inflammation. We found that EC Casp9 is implicated in contrast sensitivity decline, induction of inflammatory cytokines, and glial reactivity. One of the noted glial changes was increased levels of astroglial cl-caspase-6, which we found to be activated cell intrinsically by astroglial caspase-9 (Astro Casp9). Lastly, we discovered that Astro Casp9 contributes to capillary ischemia and contrast sensitivity decline after RVO (P-RVO). These findings reveal specific endothelial and astroglial non-apoptotic caspase-9 roles in inflammation and neurovascular injury respectively; and concomitant relevancy to contrast sensitivity decline.

    

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