Hypothermic Storage Research Articles

Nanocrystalline Cerium Dioxide Affects Erythrocyte Membrane State Under Blood Hypothermic Storage

Here, we have studied the impact of nanocrystalline cerium dioxide (NCD) particles on erythrocyte membrane state in a model of hypothermic storage of blood samples by examining the content of lipid peroxidation (LPO) primary products and hematological indices (free and total hemoglobin, hematocrit and hemolysis levels). After hypothermic storage of blood samples within 5 weeks, no significant difference in hematological indices between the control and experimental groups was revealed. In 2 weeks of hypothermic storage, the cells from experimental group in NCD presence showed an increased content of mainly those primary LPO products, which precursors were polyunsaturated fatty acids with two double bonds (conjugated dienes and oxidienes), thereby indicating the free radical processes activation. Four weeks’ storage demonstrated significantly decreased content of all primary LPO products in the experimental group cells in contrast to the control group. To the final step of the study (5 weeks of storage), the content of all primary LPO products in experimental and control groups was similar, that was an evidence of the ability of NCD particles to affect the erythrocyte membrane as well change the LPO intensity depending on storage period.

Blood Hypothermic Storage and Erythrocyte Cryopreservation in Dogs

This review presents the analysis of reported data on the current methods for blood hypothermic storage and erythrocyte cryopreservation in dogs. The relevance of designing and improving the techniques for canine erythrocyte cryopreservation, cryobanking, as well as the application of cryopreserved blood components for transfusion were substantiated. The features of canine blood hypothermic storage and specific use of cryoprotectants with various modes of their action were considered. The advantages of applying DMSO and combined cryopreservatives during low temperature storage of canine erythrocytes were specified.

Prospective In Vitro Comparison of Kerasave and Optisol-GS Corneal Storage Solutions.

The aim of this study was to compare the performance of Kerasave and Optisol-GS for hypothermic corneal storage for 14 days. This study was a prospective laboratory investigation. Mate corneas were recovered into Kerasave or Optisol-GS (27 pairs) and stored at 2°C to 8°C for 14 days. Corneas were evaluated by trained eye bank technicians, and study parameters were compared between the initial and final evaluations. Endothelial cell density (ECD), hexagonality (HEX), and coefficient of variation (CV) were evaluated by specular microscopy, and central corneal thickness (CCT) was examined by optical coherence tomography after 1, 3, 7, and 14 days of storage. Corneal transparency was scored using slit lamp examination at days 1 and 14. Average ECD, HEX, and CV for the Kerasave (2653 ± 303 cells/mm 2 , 57 ± 4%, and 36 ± 3%) and Optisol-GS (2623 ± 306 cells/mm 2 , 57 ± 5%, and 36 ± 4%) groups were not significantly different at day 1. There was also no difference at any other study time points (all P > 0.05). ECD did not significantly change from day 1 to day 14 in either group ( P > 0.05), but a statistically significant change in HEX and CV was observed between day 1 and day 14 in both groups ( P < 0.01). Average CCT measured at day 1 for corneas stored in Kerasave was 622 ± 49 μm and those stored in Optisol-GS was 580 ± 35 μm ( P < 0.01). The difference in CCT measurements was not significantly different at day 14 (Kerasave: 674 ± 46 μm vs. Optisol-GS: 647 ± 58 μm, P > 0.05). Corneal transparency was not significantly different between the 2 groups at day 1 or day 14. The corneal quality and clinically relevant parameters including ECD, endothelial morphometry, and corneal transparency were not different in corneas stored in Kerasave or Optisol-GS for 14 days. The initial difference in CCT between the 2 groups decreased at day 14. These results demonstrated that Kerasave corneal storage solution preserves the corneal endothelium similarly to Optisol-GS.

ADD10 protects renal cells from cold injuries by improving energy metabolism

Static cold storage (SCS) is currently the most widely used method for organ preservation, but a number of limitations are associated including tissue damage and restricted opportunity for organ repair. Thus, the development of improved hypothermic storage solutions is an urgent need. Herein, using a renal epithelial cell model (LLC-PK1), we tested the benefits of ADD10, a novel clinical grade antioxidant product, in reducing damages associated with ischemia-reperfusion (IR). Cells were stored up to 24h at 4 °C in University of Wisconsin (UW) solution without or in the presence of 1% ADD10 with following reperfusion up to 24h at 37 °C. The presence of ADD10 significantly decreased cells damages, cell death, and the level of reactive oxygen species (ROS) (P < 0.05). Concomitantly, ADD10 supplementation also favored an increased oxygen consumption rate (OCR) and improved bioenergetics of LLC-PK1 cells (P < 0.05). Finally, preliminary in vivo studies suggested a benefit of ADD10 on the renal function post-transplantation. In conclusion, these results demonstrate that the addition of ADD10 to the preservation solution not only efficiently protects renal cells during SCS, but also improves the functionality of cold-stored organs during transplantation.

Influence of Custodiol on Preservation of the Isolated Rat Heart during Hypothermic Storage in a High-Pressure Gas Mixture of Carbon Monoxide and Oxygen

Influence of Custodiol on Preservation of the Isolated Rat Heart during Hypothermic Storage in a High-Pressure Gas Mixture of Carbon Monoxide and Oxygen

Supplementation with uric and ascorbic acid protects stored red blood cells through enhancement of non-enzymatic antioxidant activity and metabolic rewiring

Redox imbalance and oxidative stress have emerged as generative causes of the structural and functional degradation of red blood cells (RBC) that happens during their hypothermic storage at blood banks. The aim of the present study was to examine whether the antioxidant enhancement of stored RBC units following uric (UA) and/or ascorbic acid (AA) supplementation can improve their storability as well as post-transfusion phenotypes and recovery by using in vitro and animal models, respectively. For this purpose, 34 leukoreduced CPD/SAGM RBC units were aseptically split in 4 satellite units each. UA, AA or their mixture were added in the three of them, while the fourth was used as control. Hemolysis as well as redox and metabolic parameters were studied in RBC units throughout storage. The addition of antioxidants maintained the quality parameters of stored RBCs, (e.g., hemolysis, calcium homeostasis) and furthermore, shielded them against oxidative defects by boosting extracellular and intracellular (e.g., reduced glutathione; GSH) antioxidant powers. Higher levels of GSH seemed to be obtained through distinct metabolic rewiring in the modified units: methionine-cysteine metabolism in UA samples and glutamine production in the other two groups. Oxidatively-induced hemolysis, reactive oxygen species accumulation and membrane lipid peroxidation were lower in all modifications compared to controls. Moreover, denatured/oxidized Hb binding to the membrane was minor, especially in the AA and mix treatments during middle storage. The treated RBC were able to cope against pro-oxidant triggers when found in a recipient mimicking environment in vitro, and retain control levels of 24h recovery in mice circulation. The currently presented study provides (a) a detailed picture of the effect of UA/AA administration upon stored RBCs and (b) insight into the differential metabolic rewiring when distinct antioxidant “enhancers” are used.

Swept-source OCT for corneal graft quantitative evaluation in the eye bank and the correlation of the measurements to pre-excision values

Quantitative evaluation of the human corneal grafts stored in the tissue banks is usually limited to endothelial cell density and central thickness. Swept-source OCT (SS-OCT) is capable of measuring the central curvatures of the corneal tissue prepared for transplantation without loss of sterileness, providing insights on its refractive state. The aim of the paper is to compare in vitro SS-OCT measurements with pre-excision values. Hand-held keratometry and ultrasound pachymetry was performed on 22 corneas before excision of corneoscleral button and insertion in the vial with Eusol-C solution (AlchimiaS.r.l, Nicolò, Italy). After 12 to 36 h of hypothermic storage the corneas were examined within the vials with custom built SS-OCT system maintaining a sterile environment. The anterior and posterior central curvatures, and central corneal thickness (CCT) were measured. Rotation of the corneoscleral button was controlled by making a 6-o'clock mark during excision. Mean pre-excision CCT was 626.45 ± 28.71 µm and 468.05 ± 52.96 µm when measured with SS OCT (r = 0.55; p < 0.001). Respective values for average keratometry were 7.74 ± 0.39 mm and 7.92 ± 0.57 mm (r = 0.6; p = 0.22). Although high differences were observed in corneal thickness, keratometric radius of curvature at the flat (r = 0.42; p < 0.001) and steep (r = 0.62; p = 0.014) meridian of the anterior corneal surface, as well as corneal anterior astigmatism (r = 0.3; p < 0.001), showed good correlation with pre-excision values. SS-OCT is capable of providing quantitative evaluation of the human corneal grafts in hypothermic storage. Good correlation between curvature measurements before excision and during banking in the vial indicates its clinical utility.

Long-term and short-term preservation strategies for tissue engineering and regenerative medicine products: state of the art and emerging trends.

There is an ever-growing need of human tissues and organs for transplantation. However, the availability of such tissues and organs is insufficient by a large margin, which is a huge medical and societal problem. Tissue engineering and regenerative medicine (TERM) represent potential solutions to this issue and have therefore been attracting increased interest from researchers and clinicians alike. But the successful large-scale clinical deployment of TERM products critically depends on the development of efficient preservation methodologies. The existing preservation approaches such as slow freezing, vitrification, dry state preservation, and hypothermic and normothermic storage all have issues that somehow limit the biomedical applications of TERM products. In this review, the principles and application of these approaches will be summarized, highlighting their advantages and limitations in the context of TERM products preservation.

337.2: Comparison of Hypothermic Static and Normothermic Ex-situ Donor Heart Preservation in Heterotopic Heart Transplantation With the Murine Model

Introduction and Background: Hypothermic static and Normothermic ex-situ preservation methods are the most widely used preservation technique worldwide. While cold static storage is associated with subsequent ischemic injury, Normothermic perfusion preservation has the potential to elongate preservation time with less ischemia. The current study compares the hypothermic and normothermic preservation methods in terms of graft performance, morphologic changes, and acute immune response in an experimental model. Materials and Method: Twenty-five rats underwent heterotopic abdominal heart transplantation after two hours of donor heart hypothermic storage (HT group; n=10) or normothermic ex-situ preservation (NT group; n=15). Blood samples were obtained just before and after 4-hours of implantation to analyze surface markers of immune cells (CD4, CD8, CD161, CD45R). ECG and Echocardiography were performed before harvesting and after implantation. Hearts were extracted after four hours of implantation for Hematoxylin-Eosin (HE) and TUNEL-staining, and recipient rats were euthanized. Results: 19 (76%) rats successfully survived after implantation (HT group: 100%, NT group: 60%). The mean ischemic time of the donor heart was 162.7±8.34 minutes in the HT group and 45±3.7 minutes in the NT group. The NT group showed no significant change in the heart rate before and after implantation but significantly decreased in the first hour of implantation in the HT group (259.5±24.3rpm) and normalized at 3 hours (338.3±34.1rpm). Ejection fraction and fractional shortening significantly decreased after implantation in both groups but were less significant in the NT group (P=0.001). Granulocyte was less significantly increased in the NT group compared to the HT group after the experiment (p=0.037). Although the gross structure was well preserved in both groups in HE-staining, the number of TUNEL positive cells was significantly higher in the HT group. Conclusions: Our findings suggest that normothermic ex-situ preservation is associated with well-preserved donor hearts but similar recipient immune response in comparison with hypothermic static preservation.

Osmotic Characteristics of Erythrocytes After Freezing with Composition-Modified Cryopreservatives

Here, we have studied the osmotic parameters of erythrocytes frozen in the composition-modified media with glycerol and 1,2-propanediol. A decreased glycerol concentration in a cryopreservative and an increased content of impermeable protective component sorbitol were established to affect the erythrocyte characteristics at different stages of freezing and hypothermic storage. Reduction of 1,2-propanediol and NaCl concentrations when augmenting the sucrose content in cryopreservative agent enabled changing a two-stage mode of erythrocyte freeze-thawing to the single-stage one. The presented here modification of cryoprotective medium allowed to diminish the level of damage to erythrocytes during freeze-thawing-washing out, that might prevent the inflammation development in a body when transfusing these cells.

Hypothermic Storage of 3D Cultured Multipotent Mesenchymal Stromal Cells for Regenerative Medicine Applications.

The regulatory requirements in cell processing, in the choice of a biomaterial scaffold and in quality control analysis, have to be followed in the clinical application of tissue-engineered grafts. Confirmation of sterility during quality control studies requires prolonged storage of the cell-based construct. After storage, preservation of the functional properties of the cells is an important prerequisite if the cells are to be used for cell-based tissue therapies. The study presented here shows the generation of 3D constructs based on Wharton’s jelly multipotent mesenchymal stromal cells (WJ-MSCs) and the clinically-acceptable HyaloFast® scaffold, and the effect of two- and six-day hypothermic storage of 3D cell-based constructs on the functional properties of populated cells. To study the viability, growth, gene expression, and paracrine secretion of WJ-MSCs within the scaffolds before and after storage, xeno-free culture conditions, metabolic, qPCR, and multiplex assays were applied. The WJ-MSCs adhered and proliferated within the 3D HyaloFast®. Our results show different viability of the cells after the 3D constructs have been stored under mild (25 °C) or strong (4 °C) hypothermia. At 4 °C, the significant decrease of metabolic activity of WJ-MSCs was detected after 2 days of storage, with almost complete cell loss after 6 days. In mild hypothermia (25 °C) the decrease in metabolic activity was less remarkable, confirming the suitability of these conditions for cell preservation in 3D environment. The significant changes were detected in gene expression and in the paracrine secretion profile after 2 and 6 days of storage at 25 °C. The results presented in this study are important for the rapid transfer of tissue engineering approaches into clinical applications.

Effects of prolonged cold ischemia on the DCD kidney function and Inflammasome expression in rat kidney transplants

BackgroundAcute kidney injury (AKI) is the main reason for the bad outcome of the donation of circulatory death (DCD) kidney after transplantation. Prolonged cold storage (CS) is a risk factor for the occurrence of the delayed graft function in DCD kidney. The protein NLR-domain containing receptor 3 (NLRP3) plays a crucial role in renal ischemia reperfusion injury by triggering inflammasome formation. Herein, we investigated whether the NLRP3 signal participate in the CS-induced damage of DCD kidney in rat kidney transplantation models. Materials and methodsDCD kidney and living donor (LD) kidney of SD rats were preserved in UW solution at 4 °C for 2 h or 18 h, and then transplanted into syngeneic recipient. Thus, the animals were randomly divided into 4 groups: 2-h LD group, 2-h DCD group, 18-h LD group and 18-h DCD group. The renal function and pathological changes were determined. The expressions of NLRP3 and inflammatory factor IL-1β were assessed. The concentration of ferrous iron (Fe2+) was analyzed both in kidneys and in the preservation solution. The renal morphological changes were examined by hematoxylin eosin staining. ResultsOur results showed that the levels of Cr and BUN were higher in 18-h LD group as compared to the 2-h LD group, which were remarkably increased in 18-h DCD group. The expression levels of NLRP3 and IL-1β were increased by 18-h CS compared to 2-h CS in both LD kidney and DCD kidney. In addition, the Fe2+ concentration has significantly increased in 18-h LD group than that in 2-h LD group, and the elevation of Fe2+ was more remarkable in DCD kidneys. ConclusionIn conclusion, our study demonstrated that prolonged hypothermic storage of DCD kidney deteriorated the graft function via the increased Fe2+ concentration, which was associated with the upregulation of NLRP3 expression.

Marginal Cardiac Donors and Transplant Outcomes: An 11-Year Experience

<h3>Purpose</h3> Heart transplantation is the gold standard treatment for end-stage heart failure. However, the number of available organs is numerically inadequate to address this problem. One potential way to overcome the organ shortage is to broaden criteria for acceptable donations to include a sub-pool of marginal donors. This study aims to assess our outcomes for patients receiving marginal cardiac transplants compared to their counterparts. The analysis is particularly important as our centre has recently begun to utilise the XVIVO hypothermic perfusion machine for organ storage, challenging accepted definitions of organ marginality based on ischaemic time. <h3>Methods</h3> We conducted a single-centre retrospective cohort study to identify all heart transplant recipients from 2010-2021. Patients who received a combined heart-lung transplant were excluded, as were organs obtained through the donation after circulatory death (DCD) pathway. We identified all marginal donors meeting any of the accepted criteria; donor age >60 years, donor LVEF <55%, use of T3 in the donor, or donor ischemic time >6hrs or >4hrs if donor age >40 years. <h3>Results</h3> 371 patients met our inclusion criteria for this study. Based on accepted criteria, 120 (32%) of donors were identified as marginal. Of these 120 marginal donors, 64 (53%) met the prolonged ischaemic time criteria. Overall mean donor age was 37.2 years +/- 12.6 and mean donor ischaemic time was 225 minutes +/- 67 minutes. Utilisation of extra-corporeal membrane oxygenation (ECMO) was higher in the marginal donor group (33 patients, 28%) compared to the non-marginal group (35 patients, 14%), p=0.002. There was no difference in new renal failure, requirement for dialysis or post-operative length of stay. 14 patients (6%) in the non-marginal group died within 30-days of their transplant, compared to 5 patients (4%) in the marginal group, p=0.56). <h3>Conclusion</h3> Our centre's experience indicates that carefully selected marginal donors demonstrate similar short term post-transplant outcomes when compared to their non-marginal counterparts. Furthermore, our utilisation of the XVIVO hypothermic perfusion organ storage device is likely to further challenge the accepted definition of marginal cardiac donors by allowing for longer periods of donor ischaemic time and may improve post-operative ECMO rates in the marginal donor group.

Report of the GUARDIAN-LUNG Registry: An Analysis of Advanced Hypothermic Preservation on Lung Transplantation

Purpose To assess the safety and efficacy of the LUNGguard Donor Lung Preservation System compared to conventional ice storages on lung transplant recipient perioperative and short-term clinical outcomes. Methods The Global Utilization And Registry Database for Improved preservAtion of doNor LUNGs (GUARDIAN-LUNG) study is a multi-center retrospective-prospective registry assessing clinical outcomes after lung transplantation. This is the initial report of a study comparing two methodologies of hypothermic storage: patients with donor lungs preserved by the LUNGguard and patients with donor lungs transported by conventional preservation methods (ice protocol). Data on baseline donor, recipient, and procedure characteristics are captured and reported using descriptive statistics for the two cohorts. Propensity score matching between cohorts to control for bias will be utilized to compare outcomes. Endpoints Comparisons will be made on peri-operative and short-term patient outcomes including Primary Graft Dysfunction Grade 3 during the first 72 hours, posttransplant ECMO, infections requiring treatment during initial hospitalization, and 90-day/index hospitalization mortality rates. To assess the safety and efficacy of the LUNGguard Donor Lung Preservation System compared to conventional ice storages on lung transplant recipient perioperative and short-term clinical outcomes. The Global Utilization And Registry Database for Improved preservAtion of doNor LUNGs (GUARDIAN-LUNG) study is a multi-center retrospective-prospective registry assessing clinical outcomes after lung transplantation. This is the initial report of a study comparing two methodologies of hypothermic storage: patients with donor lungs preserved by the LUNGguard and patients with donor lungs transported by conventional preservation methods (ice protocol). Data on baseline donor, recipient, and procedure characteristics are captured and reported using descriptive statistics for the two cohorts. Propensity score matching between cohorts to control for bias will be utilized to compare outcomes. Comparisons will be made on peri-operative and short-term patient outcomes including Primary Graft Dysfunction Grade 3 during the first 72 hours, posttransplant ECMO, infections requiring treatment during initial hospitalization, and 90-day/index hospitalization mortality rates.

Second Report of the GUARDIAN Registry: An International Consortium Examining the Effect of Controlled Hypothermic Preservation in Heart Transplantation

<h3>Purpose</h3> To assess the therapeutic effect of advanced organ preservation technology compared to conventional methods on heart transplant recipient perioperative and short-term clinical outcomes. <h3>Methods</h3> The Global Utilization And Registry Database for Improved heArt preservatioN (GUARDIAN) study is an international multi-center retrospective-prospective registry assessing clinical outcomes after heart transplantation. This is the second annual report of the multi-center study comparing two methodologies of hypothermic storage: patients with donor hearts preserved by advanced organ preservation [Paragonix SherpaPak Cardiac Transport System], and patients with donor hearts transported by conventional preservation methods (ice protocol). This analysis will be based on a data freeze of the registry in January 2022, which is projected to include data on over 1,000 heart transplant outcomes from 14 international centers. This will be the largest dataset on organ preservation technologies to be presented to date. Data on baseline donor, recipient, and procedure characteristics are captured to compare baseline characteristics of the two cohorts. <h3>Endpoints</h3> Comparisons will be made on peri-operative and short-term patient outcomes including hemodynamic characteristics, required surgical and medical interventions, Primary Graft Dysfunction, and length-of-stay data. Previous reports from this database have demonstrated statistically significant reductions in ICU length of stay despite longer ischemic times.

Opportunities of Electron Microscopy When Solving Cryobiological Tasks. Retrospective Analysis

The history of the development and use of low-temperature electron microscopic methods of freeze-fracture, freeze-substitution and others in cryobiological research at the Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine is presented in this article. These methods’ possibilities in studying the processes of crystal formation in cryoprotectant solutions, cell suspensions, and tissues under various freezing conditions are demonstrated. Some results of the analysis of ultrastructural changes in biological systems of various organization levels, at different stages of ontogenesis, under the influence of cooling in a wide temperature range (from 37 to –196°C) are presented. The use of a high resolution electron microscopic method in combination with an accessory technical equipment and some methodological techniques allowed to obtain fundamental results important for cryobiology on ice crystals formation and localization in the intracellular volume, the temperature-dependent transmembrane proteins redistribution, changes in the ultrastructure of erythrocytes and their membranes during hypothermic storage.

Hypothermic circulatory arrest does not induce coagulopathy in vitro

Hypothermic circulatory arrest (HCA) is an essential procedure during aortic surgery to protect organs; however, hypothermia is believed to cause coagulopathy, which is a major fatal complication. This study aimed to clarify the impact of hypothermia on coagulation by eliminating clinical biases in vitro. In the hypothermic storage study, blood samples from five healthy volunteers were stored at 37 ℃ (group N) for 3 h or at 20 ℃ for 2 h, followed by 1 h of rewarming at 37 ℃ (group H). Thromboelastography was performed before and after 3 h of storage. In the mock circulation loop (MCL) study, blood samples were placed in the MCL and (a) maintained at 37 ℃ for 4 h (group N, n = 5), or (b) cooled to 20 ℃ to simulate HCA with a 0.1 L/min flow rate for 3 h and then rewarmed to 37 ℃ (group H, n = 5). The total MCL duration was 4 h, and the flow rate was maintained at 1 L/min, except during HCA. Blood samples collected 15 min after the beginning and end of MCL were subjected to standard laboratory tests and rotational thromboelastometry analyses. Hypothermia had no impact on coagulation in both the hypothermic storage and MCL studies. MCL significantly decreased the platelet counts and clot elasticity in the INTEM and EXTEM assays; however, there was no effect on fibrinogen contribution measured by FIBTEM. Hypothermia does not cause irreversible coagulopathy in vitro; however, MCL decreases coagulation due to the deterioration of platelets.

Changes in Quality Characteristics of Crassostrea gigas Oysters during Hypothermic Storage

Now oysters are one of the most common representatives of aquaculture grown worldwide. Finding reliable ways to assess the quality of oysters is an important task for breeding professionals and users of these products. The Food and Agriculture Organization considers microbiological testing and organoleptic parameters as mandatory tests for evaluation of the mollusks’ safety. We believe these parameters in oysters are insufficient for unbiased evaluation of the condition of mollusks. In this research, we attempted to determine the most sensitive indices of oysters’ viability during hypothermic storage under anoxia conditions. We investigated the oysters during 6 days of their storage in a household refrigerator at a temperature of 5°C. According to microbiological studies, during the experiment, we expected a rise in the total number of microorganisms. However, there was a paradoxical situation when at the end of the observations the total number of microorganisms decreased significantly, which contradicted the negative assessment of the state of oysters by organoleptic parameters. Flow cytometry studies also showed no correlation between the percentages of necrotized and apoptotic cells to organoleptic parameters. Studies have shown that on day 3 of storage, the oysters demonstrated a partial deterioration in organoleptic characteristics, which is clearly reflected in the content of volatile basic nitrogen in the tissues of mollusks, which characterizes the protein breakdown and accumulation of toxic nitrogen compounds. Thus, the content of volatile basic nitrogen in the tissues of oysters is the most sensitive and significant parameter in terms of biosafety of mollusks.&#x0D; Keywords: hypothermic storage, oysters, microbial flora, food quality, nitrogen

Tangential flow filtration facilitated washing of human red blood cells: A proof-of-concept study.

Red blood cell (RBC) units in hypothermic storage degrade over time, commonly known as the RBC storage lesion. These older RBC units can cause adverse clinical effects when transfused, as older RBCs in the unit lyse and release cell-free haemoglobin (Hb), a potent vasodilator that can elicit vasoconstriction, systemic hypertension and oxidative tissue injury after transfusion. In this study, we examined a novel method of washing ex vivo stored single RBC units to remove accumulated cellular waste, specifically cell-free Hb, using tangential flow filtration (TFF) driven by a centrifugal pump. The TFF RBC washing system was run under hypothermic conditions at 4°C, at a constant system volume with 0.9wt% saline as the wash solution. The RBC washing process was conducted on 10 separate RBC units. For this proof-of-concept study, RBC units were expired at the time of washing (60-70 days old). Cell-free Hb was quantified by UV-visible absorbance spectroscopy and analysed via the Winterbourn equations. Pre- and post-wash RBC samples were analysed by Hemox Analyser, Coulter counter and Brookfield rheometer. The RBC volume fraction in solution was measured throughout the wash process by standard haematocrit (HCT) analysis. No substantial decrease in the HCT was observed during the TFF RBC washing process. However, there was a significant decrease in RBC concentration in the first half of the TFF RBC wash process, with no significant change in RBC concentration during the second half of the TFF cell wash process with an 87% overall cell recovery compared with the total number of cells before initiation of cell washing. Utilization of the extinction coefficients and characteristic peaks of each Hb species potentially present in solution was quantified by Winterbourn analysis on retentate and permeate samples for each diacycle to quantify Hb concentration during the washing process. Significant cell-free Hb reduction was observed within the first four diacycles with a starting cell-free Hb concentration in the RBC unit of 0.105 mM, which plateaus to a constant Hb concentration of 0.01 mM or a total extracellular Hb mass of 0.2g in the resultant washed unit. The oxygen equilibrium curve showed a significant decrease in P50 between the initial and final RBC sample cell wash with an initial P50 of 15.6 ± 1.8 mm Hg and a final P50 of 14 ± 1.62 mm Hg. Cooperativity increased after washing from an initial Hill coefficient of 2.37 ± 0.19 compared with a final value of 2.52 ± 0.12. Overall, this study investigated the proof-of-concept use of TFF for washing single RBC units with an emphasis on the removal of cell-free Hb from the unit. Compared with traditional cell washing procedures, the designed system was able to more efficiently remove extracellular Hb but resulted in longer wash times. For a more complete investigation of the TFF RBC washing process, further work should be done to investigate the effects of RBC unit storage after washing. The designed system is lightweight and transportable with the ability to maintain sterility between uses, providing a potential option for bedside ex vivo transfusion in clinical applications.

Proline-based solution maintains cell viability and stemness of canine adipose-derived mesenchymal stem cells after hypothermic storage.

Transportation of mesenchymal stem cells (MSCs) under hypothermic conditions in 0.9% normal saline solution (NSS) might increase cell death and alter the stemness of MSCs. The present study aimed to evaluate the effect of proline-based solution (PL-BS) on cell viability and the stemness of newly established canine adipose-derived mesenchymal stem cells (cAD-MSCs) under hypothermic conditions. Characterized cAD-MSCs were stored in 1, 10, and 100 mM PL-BS or NSS at 4°C for 6, 9, and 12 hours prior to an evaluation. The results demonstrated that storage in 1 mM PL-BS for 6 hours decreased cell apoptosis and proliferation ability, but improved cell viability and mitochondrial membrane potential. cAD-MSCs maintained their high expression of CD44 and CD90, but had a low expression of CD34 and MHC class II. Trilineage differentiation ability of cAD-MSCs was not affected by storage in 1 mM PL-BS. Gene expression analysis demonstrated that immunomodulatory genes, including IDO, HGF, PGE-2, and IL-6, were upregulated in cAD-MSCs stored in 1 mM PL-BS. In conclusion, PL-BS can be effectively applied for storing cAD-MSCs under hypothermic conditions. These findings provide a new solution for effective handling of cAD-MSCs which might be promising for clinical applications.