Zebrafish as a high throughput model for organ preservation and transplantation research.

Despite decades of effort, the preservation of complex organs for transplantation remains a significant barrier that exacerbates the organ shortage crisis. Progress in organ preservation research is significantly hindered by suboptimal research tools that force investigators to sacrifice translatability over throughput. For instance, simple model systems, such as single cell monolayers or co-cultures, lack native tissue structure and functional assessment, while mammalian whole organs are complex systems with confounding variables not compatible with high-throughput experimentation. In response, diverse fields and industries have bridged this experimental gap through the development of rich and robust resources for the use of zebrafish as a model organism. Through this study, we aim to demonstrate the value zebrafish pose for the fields of solid organ preservation and transplantation, especially with respect to experimental transplantation efforts. A wide array of methods were customized and validated for preservation-specific experimentation utilizing zebrafish, including the development of assays at multiple developmental stages (larvae and adult), methods for loading and unloading preservation agents, and the development of viability scores to quantify functional outcomes. Using this platform, the largest and most comprehensive screen of cryoprotectant agents (CPAs) was performed to determine their toxicity and efficiency at preserving complex organ systems using a high subzero approach called partial freezing (i.e., storage in the frozen state at -10°C). As a result, adult zebrafish cardiac function was successfully preserved after 5 days of partial freezing storage. In combination, the methods and techniques developed have the potential to drive and accelerate research in the fields of solid organ preservation and transplantation.

Zebrafish as a New Tool in Heart Preservation Research.

Heart transplantation became a reality at the end of the 1960s as a life-saving option for patients with end-stage heart failure. Static cold storage (SCS) at 4-6 °C has remained the standard for heart preservation for decades. However, SCS only allows for short-term storage that precludes optimal matching programs, requires emergency surgeries, and results in the unnecessary discard of organs. Among the alternatives seeking to extend ex vivo lifespan and mitigate the shortage of organs are sub-zero or machine perfusion modalities. Sub-zero approaches aim to prolong cold ischemia tolerance by deepening metabolic stasis, while machine perfusion aims to support metabolism through the continuous delivery of oxygen and nutrients. Each of these approaches hold promise; however, complex barriers must be overcome before their potential can be fully realized. We suggest that one barrier facing all experimental efforts to extend ex vivo lifespan are limited research tools. Mammalian models are usually the first choice due to translational aspects, yet experimentation can be restricted by expertise, time, and resources. Instead, there are instances when smaller vertebrate models, like the zebrafish, could fill critical experimental gaps in the field. Taken together, this review provides a summary of the current gold standard for heart preservation as well as new technologies in ex vivo lifespan extension. Furthermore, we describe how existing tools in zebrafish research, including isolated organ, cell specific and functional assays, as well as molecular tools, could complement and elevate heart preservation research.

Effects of N-acetylcysteine supplementation on cellular damage and oxidative stress indicators in volleyball athletes.

The purpose of this study was to evaluate the effects of N-acetylcysteine (NAC) supplementation on cellular damage and oxidative stress indicators in volleyball athletes. Twenty male volleyball athletes at national level performed a physical training session and were divided into 2 groups, which for 7 days took the placebo substance or NAC. After 7 days the athletes repeated the same training session. In both sessions, blood samples were collected 30 min before and immediately after the training session to measure cellular damage and oxidative stress markers. The main results show that, although higher concentrations of glutathione peroxidase and superoxide dismutase were observed in post-session 1 than those in postsession 2, the other markers showed an increase in antioxidant action after supplementation of NAC, once the effect of experimental conditions (P=0.030) were observed in: time effect (P<0.001) and interaction (P=0.019) for total glutathione; time effect (P<0.001) and interaction (P<0.001) for reduced glutathione; and time effect (P<0.001) for ferric-reducing antioxidant potential. The oxidant action indicated by the protein carbonyl was higher in the placebo group than in the NAC group (P=0.028), but a time effect (P<0.001) for the thiobarbituric acid reactive substances showed lower values in presession 1 than in presession 2. For the cellular damage markers, antagonistic results between markers were found. Based in the results, the supplementation of NAC during a short period was effective in reducing oxidant action and increasing antioxidant action. However, conclusive alterations in the responses of the cellular damage markers were not obtained.

Effect of Liposome Treatment on Hemorheology and Metabolic Profile of Human Red Blood Cells During Hypothermic Storage.

BACKGROUND: Ex vivo cold storage of red blood cells (RBCs) for transfusion has long been associated with hypothermic storage lesions. It has been proposed that liposomes can be used to mitigate hemorheological elements of RBC membrane storage lesions. This study aimed to determine the appropriate liposome treatment time and assess the effects of liposome treatment on RBC's hemorheological and metabolic profiles. MATERIALS AND METHODS: Unilamellar liposomes were synthesized to contain a bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):cholesterol (7:3 mol%). Packed human RBCs (n = 4) were divided into untreated control (HEPES-NaCl solution) and liposome-treated samples (2 mM DOPC liposomes) and treated at days 2, 21, and 42 of hypothermic storage. RBC quality assessment included percent hemolysis, deformability, aggregation, hematological indices, microvesiculation, supernatant potassium, adenosine triphosphate (ATP), and 2,3-diphosphoglycerate (2,3-DPG). RESULTS: Among the parameters affected by liposome treatment time were deformability, aggregation amplitude (Amp), mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and microparticle mean fluorescence intensity. After 6 weeks of storage, aggregation index (AI) and Amp were significantly increased in liposome-treated RBCs (AI: 45.38 ± 1.92% vs. 41.54 ± 4.10%, p = 0.020; Amp: 16.38 ± 2.17 arbitrary units [au] vs. 12.22 ± 3.29 au, p = 0.019). Despite comparable hemolysis levels at 3 and 6 weeks, DOPC-treated RBCs showed significantly increased potassium levels for the same time points (3 weeks: 31.2 ± 2.7 mmol/L vs. 30.8 ± 2.7 mmol/L, p = 0.007; 6 weeks: 45.0 ± 3.0 mmol/L vs. 43.8 ± 3.4 mmol/L, p = 0.013). ATP and 2,3-DPG levels were comparable throughout storage. CONCLUSIONS: Liposome treatment seemed to be more beneficial when performed at the beginning of storage up to day 21. DOPC liposome treatment resulted in an improvement in human RBC hemorheology upon storage, with no significant impact on metabolic profile.

Evaluation of the functional properties of cryopreserved buffy coat-derived monocytes for monocyte monolayer assay.

BACKGROUND: Monocyte monolayer assay (MMA) is a compatibility testing method for evaluating the clinical significance of red blood cell (RBC) alloantibodies. Time-consuming monocyte isolation procedures and requirement for fresh monocytes have limited application of the MMA. The aim of this study was to develop and assess the utility and efficacy of cryopreserved buffy coat (BC)-derived monocytes for MMA application. STUDY DESIGN AND METHODS: Peripheral blood mononuclear cells (PBMNCs) were isolated from BC or peripheral blood (PB) and pooled and BC PBMNCs were cryopreserved. Monocytes from pooled PBMNCs were incubated with anti-D-sensitized, anti-Scianna2 (Sc2)-sensitized, anti-AnWj-sensitized, or anti-Jra -sensitized RBCs or lipopolysaccharide (LPS). MMA phagocytic index (PI) and membrane integrity were determined microscopically, and cytokine release was measured by Luminex technology. RESULTS: PBMNC isolation rates from fresh BC and PB were not comparable (67.4 ± 6.3 and 75.8 ± 7.7% respectively, p = 0.024). There was no significant difference in PBMNC membrane integrity (fresh PB, 100%; fresh BC, 100%; cryopreserved BC, 95.2 ± 1.2%), postwash recovery (fresh PB, 85.9 ± 3.1; fresh BC, 86.9 ± 6.7; cryopreserved BC, 84.8 ± 5.1), or monocyte PI (fresh PB, 82 ± 10; fresh BC, 77 ± 11; cryopreserved BC = 80 ± 6). Monocytes from pooled cryopreserved BC PBMNCs reacted with RBCs sensitized with anti-D and RBC alloantibodies, including anti-Sc2, anti-Jra , and anti-AnWj. CONCLUSIONS: Monocytes from pooled cryopreserved BC PBMNCs can be used reliably to evaluate phagocytic responses of sensitized RBCs and to assess clinical significance of RBC alloantibodies.

The immune-stimulation capacity of liposome-treated red blood cells.

Our in vivo studies on a rat model established safety of transfusing liposome-treated red blood cells (RBCs) but identified the potential for immune modulation as related to transfusion efficacy of liposome-treated RBCs. The aim of this study was at assessing the impact of liposome-induced membrane changes on the immune profile of liposome-treated RBCs by (a) evaluating their interaction with endothelial cells and monocytes; and (b) the resulting immune response derived from this interaction, in the form of cytokine release, adhesion molecules expression and phagocytosis. Unilamellar liposomes were synthesized to contain unsaturated phospholipids (1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC]:CHOL, 7:3 mol%). The human RBCs immune profile was assessed by incubating control and DOPC-treated RBCs with human umbilical vein endothelial cells (HUVECs) and monocytes. Cytokine release measured by Luminex technology, vascular cell adhesion molecule (VCAM)-1 and E-selectin on HUVECs measured by flow cytometry, and the erythrophagocytic activity of monocytes by monocyte monolayer assay (MMA) were determined. Fibroblast growth factor [FGF]-2 was the only cytokine released by HUVECs that remained increased after incubation with DOPC-treated RBCs compared to control throughout storage. The expression of both VCAM-1 (15.3 ± 5.6% versus 6.3 ± 0.9%, p = 0.008) and E-selectin (18.0 ± 6.3% versus 6.6 ± 0.7%, p = 0.004) by HUVECs were significantly increased after incubation with DOPC-treated RBCs at day 2 of storage. The MMA resulted in phagocytic indexes of zero for both control and DOPC-treated RBCs at day 2 and 42 of storage. The liposome treatment did not result in significant changes to the immune profile of stored DOPC-treated RBCs. These findings combined with previous in vivo results, make liposome treatment a potential candidate for application in RBC preservation and open the possibility for clinical use with other cell types.

Effect of liposome-treated red blood cells in an anemic rat model.

CONTEXT: Liposomes have been shown to improve human red blood cell (RBC) in vitro quality by minimizing membrane damage occurring during 42-d hypothermic storage. Small animal models are necessary to evaluate novel blood products and guide future clinical studies. OBJECTIVES: The aim of this study was to assess the effect of liposome treatments on rat RBC hypothermic storage lesion (HSL) and to examine in vivo outcomes of transfusing liposome treated RBCs in a rat model. MATERIALS AND METHODS: Unilamellar liposomes were synthesized which contained saturated (DPPC:CHOL, 7:3 mol%), unsaturated (DOPC:CHOL, 7:3 mol%), saturated charged (DPPC:CHOL:PS, 6:3:1 mol%), and unsaturated charged (DOPC:CHOL:PS, 6:3:1 mol%) phospholipids. After liposome treatment, rat RBC quality was assessed by percent hemolysis, deformability, aggregation, hematological indices, microvesiculation, and cholesterol/phospholipid concentrations. An anemic rat model of myocardial ischemia and reperfusion (I/R) was used to evaluate the outcomes of transfusing liposome-treated RBCs. RESULTS: All four liposome treatments resulted in significant decreases in hemolysis, with the most prominent effect seen with DOPC-liposomes (DOPC: 1.6 ± 0.1% versus control: 3.1 ± 0.2%, p = 0.015). RBCs treated with uncharged liposomes had lower hemolysis compared with charged liposomes (3.4 ± 0.2% versus 3.9 ± 0.4%, p = 0.010). The in vivo study showed no significant difference in the hemoglobin levels and infarct size (53.3 ± 13.1% versus 45.3 ± 8.4%, p = 0.223) between liposome and control groups. DISCUSSION AND CONCLUSION: Liposome treatment improved in vitro quality of stored rat RBCs. However, the changes observed in vitro were not sufficient to improve the in vivo outcomes of myocardial I/R in anemic rats transfused with liposome-treated RBCs.