Engineered Supercooling Systems for Enhanced Long-Term Preservation of Large-Volume Red Blood Cells in Commercial Blood Bags

Engineered Supercooling Systems for Enhanced Long-Term Preservation of Large-Volume Red Blood Cells in Commercial Blood Bags | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Engineered Supercooling Systems for Enhanced Long-Term Preservation of Large-Volume Red Blood Cells in Commercial Blood Bags Qi Liu, Shichun Wang, Jie Yan, Ronghua Diao, Haishui Huang, Feng Xu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6029466/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Extending the shelf life of red blood cells (RBCs) is vital for transfusion medicine, yet traditional storage methods like refrigeration and cryopreservation suffer from limitations such as storage lesions and ice-induced damage. We developed an improved supercooling preservation system for large-volume (100 ml) RBC suspensions in commercial polyvinylchloride (PVC) blood bags by minimizing favorable sites of ice nucleation and maintaining precise thermal control at − 8°C. This engineered protocol significantly reduces hemolysis, metabolic degradation, and oxidative stress while preserving RBC membrane integrity and functionality for up to 63 days. In vivo transfusion studies in New Zealand white rabbits demonstrate that supercooling-preserved RBCs achieve higher post-transfusion recovery rates, outperforming conventional storage methods. Our scalable and cost-effective supercooling system integrates seamlessly with existing blood banking infrastructure, addressing critical needs for extended RBC storage and improved transfusion outcomes. This advancement enhances blood supply reliability and patient care, representing a significant breakthrough in transfusion medicine. Supercooling Preservation Red Blood Cells Blood Bags Metabolic Stability Oxidative Stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Red blood cell (RBC) transfusion is one of the most common and critical medical interventions worldwide, essential for managing conditions such as acute hemorrhage, chronic anemia, and supporting surgical procedures. [ 1 – 3 ] The yearly donation of 120 million blood units globally, yet many regions continue to face significant disparities between blood supply and clinical demand. [ 4 ] Effective storage and preservation of RBCs are crucial to ensuring a stable and safe blood supply, directly impacting patient care and transfusion outcomes. [ 5 – 7 ] Currently, RBC suspensions are stored at 4°C in polyvinylchloride (PVC) blood bags for up to 42 days. [ 8 ] However, RBC units experience "storage lesions" within two weeks at hypothermic conditions. These storage lesions include reduced levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG), increased oxidative stress, and membrane protein alterations, all of which compromise RBC viability and efficacy. [ 9 – 13 ] Clinical studies have linked the transfusion of older RBC units (> 2 weeks) to elevated risks of postoperative complications, infections, and higher mortality rates. [ 14 ] While cryopreservation extends RBC shelf life beyond a decade using high concentrations of glycerol (> 20%) as a cryoprotective agent, it introduces significant challenges. [ 15 ] The addition and removal of glycerol cause osmotic stress and hemolysis, and the deglycerolization process is laborious and time-consuming, delaying emergency transfusions. [ 16 , 17 ] Additionally, cryopreservation risks cryoinjuries such as intracellular ice formation and recrystallization, further reducing RBC viability. [ 18 , 19 ] Alternative methods like freeze-drying, though promising for room-temperature storage, still expose RBCs to ice-induced damage, resulting in reduced viability. [ 20 ] Recent advancements have explored novel cryoprotectants (e.g., trehalose, tricine, nanomaterials) and innovative storage technologies (e.g., anaerobic storage, hydrogel encapsulation). [ 21 – 26 ] Despite promising laboratory results, these methods often involve complex procedures, severe hemolysis, small volume, low recovery rates, and lack scalability for clinical application. Supercooling has emerged as a transformative approach, maintaining RBCs below their freezing point without ice formation, typically between − 20°C and − 3°C. [ 27 – 33 ] This method minimizes ice-mediated damage and metabolic activity, preserving cell integrity over extended periods. Previous studies have successfully supercooled small volumes (1 ml) of RBC suspensions in polystyrene rigid tubes for up to 100 days using paraffin oil sealing, achieving minimal hemolysis and high hemoglobin recovery rates. [ 34 ] However, supercooling preservation is still limited to small-volume storage and is insufficient to meet the requirements for large-volume RBC preservation in clinical settings. Currently, maintaining adequate RBC supplies in blood banks relies on advanced storage systems utilizing commercial blood bags constructed from flexible, sterile, gas-permeable PVC materials. These commercial blood bags preserve cellular integrity through three key mechanisms: 1) minimizing mechanical hemolysis via compliant and super smooth surfaces, 2) facilitating essential gas exchange through selective membrane permeability, and 3) maintaining metabolic homeostasis and reducing oxidative stress through optimal additive solutions. This multimodal preservation approach significantly enhances transfusion safety while complying with international blood banking gold standards (AABB guidelines). [ 14 ] However, scaling supercooling preservation techniques to clinically large-volume at physiological hematocrit levels presents below technical challenges. Increased ice nucleation propensity governed by the classical nucleation theory relationship, where larger volumes and extended storage durations exponentially raise crystallization probability. Mechanical instability of PVC bags under subzero temperatures, potentially compromising bag integrity during phase change events. [ 18 , 35 – 37 ] This thermodynamic-structural paradox creates critical barriers to implementing supercooling preservation in standard blood banking operations, necessitating fundamental material science innovations coupled with advanced thermal control strategies. In this study, we present an innovative supercooling preservation protocol tailored for large-volume (100 ml) RBC suspensions stored in commercial PVC blood bags. By engineering a system that minimizes nucleation sites through paraffin oil sealing and stabilizes storage conditions with precise thermal control, we achieved successful supercooling of RBCs at -8℃ for up to 63 days under meet the Food and Drug Administration (FDA) standards condition. Compared to traditional supercooling preservation methods, we can now increase the RBC storage volume by up to 100 times, maintaining a standard hematocrit level, all while employing a standard blood bag as the storage container. Our protocol significantly reduces hemolysis, metabolic degradation, and oxidative stress while maintaining RBC membrane integrity and functionality. In vivo transfusion studies in New Zealand white rabbits demonstrated superior post-transfusion recovery rates compared to conventionally stored RBCs. This scalable and cost-effective supercooling system in commercial PVC blood bags is compatible with existing blood banking infrastructure, offering a robust and clinically relevant solution poised to transform transfusion medicine by extending RBC shelf life and enhancing transfusion outcomes. 2 Results 2.1 Optimization of supercooling preservation conditions enhances RBC stability. Supercooling preservation relies on preventing heterogeneous nucleation at the liquid-air interface, typically achieved using an immiscible oil phase. [33, 34, 38] While this strategy has proven effective in rigid round-bottomed polystyrene tubes, which maintain the stability of the oil-water interface, its application in flexible PVC blood bags presents significant challenges. Flexible blood bags are prone to deformation under external forces, potentially disrupting the oil layer and initiating ice nucleation, thereby compromising the supercooled state. To address this issue, we adhered the outer surface of the PVC blood bags to a rigid baseplate using double-sided tape, thereby preventing deformation and ensuring the integrity of the oil seal during storage. This engineering modification is crucial for maintaining a stable supercooled environment in flexible blood bags. The maintenance of metastable supercooled states in RBC suspensions in PVC blood bags is critically influenced by multiple factors. To develop an optimized supercooling protocol for RBC in PVC blood bags, an orthogonal experimental design (Table 1) was implemented to systematically evaluate six critical factors at each three levels: 1) paraffin oil sealing volume, 2) storage temperature, 3) RBC suspension volume, 4) cooling rate, 5) blood bag placement angle, and 6) baseplate material. The liquid-air interface is thermodynamically conducive to heterogeneous ice nucleation due to surface tension; therefore, we minimized potential ice nucleation sites by sealing the blood bags with paraffin oil. Additionally, ice formation during the supercooling process is a stochastic phenomenon influenced by several factors, including RBC volume, storage temperature, and cooling rate. The likelihood of ice nucleation within the supercooled volume increases with larger RBC volumes, faster cooling rates, and lower storage temperatures. Furthermore, the blood bag placement angle directly modulates the RBC suspension/air interfacial area and wrap-baseplate material selection significantly impacts thermal exchange dynamics ( Fig. S1 ). Table 1. Experimental factors with three levels. Level Placement angle Baseplate materials Cooling rate Storage volume Storage temperature Sealing oil volume I flat styrofoam rapid 100 ml -12°C 8 ml II tilt glass middle 150 ml -10°C 14 ml III vertical copper plate slow 200 ml -8°C 20 ml The orthogonal experimental design systematically assessed three levels for each of the six factors, reducing the complexity from 729 potential combinations to 18 experimental setups. Each setup was evaluated based on freezing frequency, defined as the ratio of frozen samples to total samples, to determine supercooling stability. Analysis of variance revealed that storage temperature (Vk = 0.1333) and RBC volume (Vk = 0.1282) were the most significant factors influencing freezing frequency, followed by placement angle (Vk = 0.0971) ( Table 2 ). Cooling rate, baseplate material, and sealing oil volume also impacted freezing frequency but to a lesser extent. Under the optimal conditions identified, i.e. , storing 100 ml of RBC suspensions sealed with 8 ml of paraffin oil, cooled slowly to -8.0°C, and maintained vertically in a monitoring refrigerator, we achieved the lowest freezing frequency, indicating enhanced supercooling stability ( Fig. S2 ). Table 2. Orthogonal experimental design results and factor significance. Test No. Placement angle Baseplate materials Cooling rate Storage volume Storage temperature Sealing oil volume Freezing frequency (F f ) Transformed F f 1 tilt − II glass − II rapid − I 150 ml − II -10℃ −II 20 ml − III 80% 1.11 2 flat − I copper − III slow − III 150 ml − II -10℃ −II 14 ml − II 70% 0.99 3 vertical − III styrofoam − I rapid − I 200 ml − III -10℃ −II 20 ml − III 70% 0.99 4 vertical − III styrofoam − I slow − III 100 ml − I -10℃ −II 14 ml − II 30% 0.58 5 tilt − II styrofoam − I middle − II 200 ml − III -8℃ −III 14 ml − II 50% 0.79 6 flat − I glass − II rapid − I 200 ml − III -8℃ −III 14 ml − II 90% 1.25 7 flat − I styrofoam − I middle − II 150 ml − II -12℃ −I 20 ml − III 80% 1.11 8 tilt − II styrofoam − I slow − III 150 ml − II -8℃ −III 8 ml − I 30% 0.58 9 flat − I glass − II slow − III 100 ml − I -8℃ −III 20 ml − III 40% 0.68 10 vertical − III copper − III middle − II 100 ml − I -8℃ −III 20 ml − III 20% 0.46 11 tilt − II copper − III rapid − I 100 ml − I -12℃ −I 14 ml − II 70% 0.99 12 tilt − II glass − II middle − II 100 ml − I -10℃ −II 8 ml − I 50% 0.79 13 vertical − III glass − II slow − III 200 ml − III -12℃ −I 8 ml − I 80% 1.11 14 vertical − III copper − III rapid − I 150 ml − II -8℃ −III 8 ml − I 30% 0.58 15 flat − I styrofoam − I rapid − I 100 ml − I -12℃ −I 8 ml − I 70% 0.99 16 vertical − III glass − II middle − II 150 ml − II -12℃ −I 14 ml − II 60% 0.89 17 flat − I copper − III middle − II 200 ml − III -10℃ −II 8 ml − I 80% 1.11 18 tilt − II copper − III slow − III 200 ml − III -12℃ −I 20 ml − III 70% 0.99 I 6.13 5.04 5.91 4.49 6.08 5.16 II 5.25 5.83 5.15 5.26 5.57 5.49 III 4.61 5.12 4.93 6.24 4.34 5.34 S k 0.1941 0.0630 0.0881 0.2564 0.2667 0.0091 V k 0.0971 0.0315 0.0441 0.1282 0.1333 0.0046 NOTE : I, II, and III are the summations of the transformed freezing frequencies from the tests that involve levels I, II, and III, respectively. The smallest values among I, II, and III indicate the level with the lowest freezing frequency for the sample. S k is the sum of the squares, which corresponds to the variation in the variety mean. V k is the mean square and is defined as V k = S k / f k , where f k is the number of degrees of freedom. V k represents the significance of each factor's main effect, and a higher V k value signifies a greater significance for the freezing frequency. By our analysis, the best combination is slow cooling, styrofoam baseplate, 8 ml of sealing oil, -8℃, vertical orientation and 100 ml storage volume. N = 10 (Each condition was tested once with 10 replicate samples). F f : Freezing frequency. Transformed F f = arcsin(\(\:\sqrt{freezing\:frequency}\)). S k =(I 2 + II 2 + III 2 ) / 6 - (I + II + III) 2 / 18; V k = S k / 2. The engineered steps for the supercooling preservation of RBC in commercial blood bags are illustrated in Fig. 1A . Supercooled state stability relies on two parameters, i.e. , slower cooling rate to the supercooling temperature and minimal temperature fluctuations during storage. [28, 39–44] However, During the blood storage process, temperature fluctuations within the blood storage refrigerator are inevitable due to its operational mode of the cooling and heating cycles. We further analyzed the impact of various thermally conductive materials (copper, glass, styrofoam) as baseplates for supercooled storage. During cooling, we recorded temperature changes at the bottom, middle, and top of the blood bags. Copper takes about 130 mins, glass 150 mins, and styrofoam 360 mins to reach the target temperature of -8.0°C ( Fig. 1B-D ). The refrigerator's cyclical freezing compressor activity causes temperature oscillations every 30 mins, with fluctuations around 2.3°C. Different wraps of the blood bag modify temperature fluctuations: 2.0°C for wrap-copper, 0.7°C for wrap-glass, and 0.3°C for wrap-styrofoam ( Fig. 1E-F ). We also assessed freezing frequencies for blood cells at various subzero storage temperatures (-8.0 to -13.0°C) preserved for 2 days. At -10.0°C, the freezing frequency is 90% for wrap-copper, 60% for wrap-glass, and 30% for wrap-styrofoam ( Fig. 2G ). Specifically, the use of styrofoam as the baseplate material supported a slower cooling rate and minimized temperature fluctuations during long-term storage, further reducing the likelihood of ice nucleation ( Fig. 1H-I ). Our results further confirm that maintaining a slow cooling rate and minimizing temperature fluctuations were critical in sustaining the supercooled state as evidenced by the superior performance of styrofoam-wrapped blood bags compared to those wrapped with copper or glass ( Fig. J ). These findings demonstrate that precise control of cooling parameters and sealing methods, combined with structural reinforcement of the blood bags, are essential for maintaining the supercooled state in large-volume RBC suspensions in commercial blood bags. The optimized protocol not only minimizes ice nucleation but also ensures scalability and reliability, making it suitable for clinical applications. By addressing the inherent challenges associated with flexible blood bags, our engineered supercooling preservation system significantly enhances RBC stability, laying the groundwork for improved long-term storage solutions in transfusion medicine. 2.2 Supercooling preservation minimizes hemolysis and metabolic degradation of RBC. To evaluate the efficacy of supercooling preservation in maintaining RBC integrity and metabolic stability, we conducted comprehensive in vitro analyses over a 63-day storage period ( Fig. 2A, S3 ). Hemolysis is a critical indicator of RBC preservation quality, with the FDA standard set at below 1%. [45] Hemolysis was assessed by measuring free hemoglobin (F-Hb) levels in the supernatant post-centrifugation. Visual inspection of RBC supernatants ( Fig. 2B ) corroborated these findings, with supercooling-preserved samples displaying less discoloration, indicative of reduced hemolysis. The supercooling group exhibits a significantly lower hemolysis rate of 0.76% at day 63 compared to the control group’s 0.88% ( p < 0.05) ( Fig. 2C ). To assess metabolic stability, we measured key parameters including glucose consumption, lactate accumulation, pH levels, adenosine triphosphate (ATP), and 2,3-diphosphoglycerate (2,3-DPG) concentrations. The supercooling group maintains significantly higher glucose levels and exhibits lower lactate accumulation compared to the control group, with notable differences at 42 days ( p = 0.002 for glucose; p < 0.05 for lactate) ( Fig. 2D-E ). Additionally, the decline in pH is less pronounced in the supercooling group, reaching 6.43 at day 63 versus 6.19 in the control group ( p < 0.05) ( Fig. 2F ). ATP levels, essential for RBC functionality, remain significantly higher in the supercooling group (3.06 nmol/mg) compared to the control group (2.17 nmol/mg) at 42 days ( p < 0.05) (Fig. 2G) . The concentration of 2,3-DPG, crucial for oxygen release capacity, shows a markedly slower decline in the supercooling group, with both groups leveling off post 42 days ( p = 0.206) ( Fig. 2H ). These results indicate that supercooling preservation effectively decelerates metabolic degradation of RBC, maintaining essential biochemical functions over extended storage durations. Collectively, these results demonstrate that supercooling preservation at -8°C significantly minimizes hemolysis and metabolic degradation of RBC. The maintained levels of ATP and 2,3-DPG, indicate that supercooling effectively preserves RBC functionality and viability over prolonged storage periods. Consequently, our supercooling protocol offers a superior preservation method, mitigating the hemolysis and metabolic lesions. 2.3 Supercooling preservation reduces oxidative stress and maintains RBC membrane integrity. To evaluate the impact of supercooling preservation on oxidative stress and membrane integrity in RBCs, we conducted a series of biochemical assays and morphological analyses over a 63-day storage period (Fig. 3A) . We measured ROS and MDA levels to assess oxidative stress, and evaluated the activities of superoxide dismutase (SOD) and catalase (CAT) to determine the RBCs' antioxidative defense capacity. Additionally, we examined membrane integrity through osmotic fragility tests and phosphatidylserine (PS) exposure, and analyzed RBC morphology using blood smears. We observed that RBCs preserved under supercooling conditions exhibit significantly lower increases in ROS and MDA levels compared to the control group stored at 4°C ( p < 0.05) (Fig. 3B-C) . Specifically, ROS levels in the supercooling group rise modestly over time, whereas the control group shows a higher increase, indicating higher oxidative stress in conventional storage. Similarly, MDA concentrations, a marker of lipid peroxidation, remain relatively stable in the supercooling group but increase significantly in the control group, reflecting reduced lipid membrane damage in supercooled RBCs. Furthermore, the activities of antioxidant enzymes SOD and CAT remain largely stable in the supercooling group throughout the storage period, with only minor decreases observed towards day 63 ( Fig. 3D-E ). In contrast, the control group exhibits a significant decline in CAT activity by the end of the storage period, suggesting compromised antioxidative defenses. These results indicate that supercooling preservation effectively mitigates oxidative stress, maintaining the antioxidative capacity of RBCs and protecting them from oxidative damage. To assess membrane integrity, we performed osmotic fragility tests and measured PS exposure on RBC surfaces. The supercooling group demonstrates lower mean cell fragility (MCF) values compared to controls, indicating enhanced membrane stability (Fig. 3F) . Flow cytometry analysis revealed significantly reduced PS exposure in the supercooling group at day 63 ( p < 0.05) (Fig. 3G, S4) , suggesting diminished membrane asymmetry disruption and delayed recognition by phagocytes. Morphological assessments using blood smears showed that RBCs in the supercooling group retain their typical elliptical, biconcave shape with minimal formation of sphero-echinocytes and fragmented cells (Fig. 3H, S5) . In contrast, control RBCs exhibit pronounced morphological abnormalities, including increased echinocytosis and cell fragmentation, indicative of membrane damage and compromised structural integrity. These findings demonstrate that supercooling preservation at -8°C effectively reduces oxidative stress and maintains RBC membrane integrity over extended storage periods. By minimizing ROS and MDA accumulation and preserving antioxidant enzyme activities, our supercooling protocol protects RBCs from oxidative damage. Additionally, the maintenance of membrane integrity and normal morphology in supercooled RBCs underscores the method's efficacy in preserving cellular functionality and viability. Consequently, supercooling presents a superior preservation strategy by mitigating oxidative stress and membrane damage associated with conventional refrigeration, thereby enhancing the overall quality and safety of stored RBCs for transfusion. 2.4 Supercooling-preserved RBCs exhibit superior in vivo transfusion quality. To evaluate the clinical viability of supercooling-preserved RBC, we conducted in vivo transfusion studies using New Zealand white rabbits. Higher percentages and longer periods of circulating RBC indicate superior RBC quality (Fig. 4A) , adhering to FDA guidelines, which define a successful transfusion as maintaining over 75% of transfused RBCs in circulation after 24 hours, [3] we assessed the recovery rates of supercooling-preserved RBCs compared to conventionally stored controls. We first isolated RBCs from rabbit whole blood by sequential centrifugation to remove platelet-rich plasma and buffy coat, followed by resuspension in mannitol–adenine–phosphate (MAP) solution. RBCs were then either stored at 4°C (control group) or subjected to our supercooling preservation protocol at − 8°C (supercooling group) for varying durations (0, 14, 21, and 35 days). At each storage interval, RBCs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE + ) to facilitate tracking post-transfusion. Approximately 4 ml of labeled RBCs were transfused into recipient rabbits via the ear vein (Fig. 4B) . Morphological assessments using blood smears revealed that supercooled RBCs of rabbit retain their typical elliptical, biconcave shape with minimal morphological abnormalities over time (Fig. 4C) , whereas control RBCs exhibit increased sphero-echinocyte and fragmented cell formations indicative of membrane damage and reduced deformability. Immediately after transfusion (0-day storage), both supercooling-preserved and control RBCs demonstrate high recovery rates, with fresh RBCs achieving a 94.83% recovery at 24 hours post-transfusion. After 14 days of storage, the control group shows an 82.69% recovery rate, while the supercooling group maintains over 90% recovery. At 21 days, recovery rates declined to 65.26% in the control group and remained robust at 84.09% in the supercooling group. Although at 35 days, the supercooling group's recovery rate decreases to 53.01%, falling below the FDA’s threshold, it was still significantly higher than the control group’s rate (Fig. 4D) . Our nonradioactive flow cytometry method, based on CFSE + labeling, demonstrated that nearly all transfused RBCs are successfully labeled, ensuring accurate tracking of RBC survival. The log fluorescence intensity versus cell counts plots confirmed consistent CFSE + staining across all samples (Fig. 4E, S6) . Supercooling-preserved RBCs showed a higher persistence of CFSE + cells in circulation at 24 hours post-transfusion compared to control RBCs, corroborating the recovery rate data. These findings indicate that supercooling preservation significantly enhances the in vivo transfusion quality of RBCs. Supercooled RBCs maintained higher recovery rates at critical storage intervals (14 and 21 days), demonstrating superior viability and functionality compared to conventionally stored controls. Although the recovery rate for supercooled RBCs decreased below the FDA threshold at 35 days, it remained markedly higher than that of the control group, highlighting the effectiveness of supercooling in preserving RBC quality over extended storage periods. The superior in vivo recovery rates of supercooling-preserved RBCs suggest enhanced transfusion outcomes, potentially reducing the risks associated with transfusion of older blood units, such as postoperative complications and increased mortality rates. This advancement underscores the translational potential of our supercooling preservation protocol, offering a viable solution to extend RBC shelf life and improve the safety and efficacy of blood transfusions in clinical settings ( Table. S1 ). 3 Discussion Traditional methods for RBC preservation, particularly cryopreservation, offer extended storage durations but are hindered by significant challenges. Cryopreservation necessitates the use of high concentrations of glycerol (> 20%) as cryoprotective agents (CPAs) to maintain RBC viability below − 65°C. [ 21 , 23 , 46 ] However, the addition and subsequent removal of glycerol induce transient osmotic gradients, resulting in cytotoxicity and osmotic injuries that lead to severe hemolysis [ 19 ] . Additionally, the deglycerolization process involves multiple washing and centrifugation steps, which can delay emergency transfusions. [ 16 , 22 , 47 , 48 ] Alternative preservation methods, such as freeze-drying, offer a promising solution for room-temperature storage of RBC by eliminating the need for ultra-low temperatures. However, this method is still limited by ice crystal formation during the freezing phase, which can cause mechanical damage to cellular membranes and hemoglobin structures. [ 20 ] These limitations underscore the urgent need for innovative preservation techniques that can extend RBC shelf life without compromising cellular integrity or clinical utility. 3.1 Engineered supercooling protocol enhances stability and scalability for large-volume RBC preservation. Our study introduces an engineered supercooling preservation protocol specifically designed to address the challenges associated with large-volume RBC storage in commercial PVC blood bags. By integrating paraffin oil sealing with precise thermal control mechanisms, we successfully maintained 100 ml of RBC suspensions at − 8°C for up to 63 days ( Fig. 1A ). The use of paraffin oil effectively eliminates heterogeneous nucleation at the liquid-air interface, a critical factor in preventing ice crystal formation in the metastable supercooled state. [ 34 ] Additionally, adhering the flexible PVC blood bags to rigid baseplates using double-sided tape minimized deformation and ensured the stability of the oil seal during storage, a key innovation that differentiates our protocol from previous approaches. [ 32 – 34 , 49 , 50 ] The orthogonal experimental design optimized six critical factors (storage temperature, RBC volume, placement angle, cooling rate, baseplate material, and sealing oil volume), resulting in enhanced supercooling stability with minimal freezing events ( Table. 2 ). Although hypothermic storage (4°C) and cryopreservation are widely utilized for RBC preservation, both approaches exhibit critical limitations that may compromise transfusion efficacy and safety. For hypothermic storage, the restricted shelf life (typically ≤ 42 days) is accompanied by a progressive decline in RBC quality. In contrast, cryopreservation extends storage duration to years but introduces technical and biological challenges [ 14 ] . One recent advance is the preservation of RBCs at -8.0°C for an impressive 70 days without freezing by adding a low concentration of glycerol combined with PEG-400 to reduce the freezing point. However, given the tedious rewarming and washing processes and very small storage volume (5 ml), its clinical practicality remains limited [ 46 ] . Our engineering strategy not only ensures the reliability and reproducibility of the supercooling state in large-volume suspensions in commercial blood bags but also highlights the scalability and practicality of our preservation system for clinical applications ( Fig. 5 ). 3.2 Supercooling mitigates metabolic and oxidative stress, preserving RBC functionality. Supercooling preservation significantly mitigates metabolic and oxidative stress of RBC, thereby maintaining cellular functionality and viability over extended storage periods. Our results demonstrate that supercooled RBCs exhibit reduced hemolysis (0.76% in supercooling vs 0.88% in controls after 63 days) and maintain higher levels of essential metabolic markers such as glucose, ATP, and 2,3-diphosphoglycerate (2,3-DPG) compared to conventionally stored RBCs. The slower glycolysis rate in supercooled RBCs leads to delayed lactate accumulation and less pronounced pH drops, indicating a deceleration of metabolic degradation. [ 51 ] Oxidative stress was an important mechanism of injury at subzero temperatures. Blood bags, optimize oxygen and nutrient exchange due to gas-permeable surfaces, provide a more controlled oxygen environment. In addition, the choice of an appropriate additive solution (AS) for RBC is crucial for supercooling preservation at low temperatures. [ 33 ] In this study, we selected the latest and most comprehensive formulation of mannitol-adenine-phosphate (MAP) solution as the AS for RBC storage. Compared to the other AS, the RBC stored in MAP solution experienced lower hemolysis and ensured continued glycolytic activity during supercooling preservation, primarily due to its multifaceted mechanisms in mitigating oxidative damage, stabilizing metabolic homeostasis. For example, mannitol scavenges oxygen radical via its hydroxyl groups and optimizes antioxidant capacity through the citrate-phosphate buffer system, directly attenuating oxidative stress on membrane lipids and hemoglobin [ 52 ] . By maintaining metabolic homeostasis and reducing oxidative insults, supercooling preservation preserves RBC membrane integrity and prevents morphological transformations, such as echinocytosis and cell fragmentation. These biochemical and structural protections collectively enhance the overall quality and functionality of RBCs, ensuring their efficacy for transfusion purposes. 3.3 Supercooling-preserved RBC demonstrate superior transfusion outcomes with clinical potential. The ultimate test of RBC preservation efficacy lies in their performance during transfusion. Our in vivo studies using New Zealand white rabbits demonstrate that supercooling-preserved RBCs exhibit superior post-transfusion recovery rates compared to conventionally stored controls. Following storage for 28 days, supercooled RBCs maintained recovery rates exceeding 75%, significantly outperforming the control group, which showed recovery rates of 50.13%, respectively. Although recovery rates for supercooled RBCs declined to 53.01% at 35 days, this still represented a substantial improvement over the control group (25.69%) and highlighted the potential for further optimization to meet FDA guidelines for successful transfusions. [ 53 ] Morphological analyses confirmed that supercooled RBCs retained their typical elliptical, biconcave shape with minimal morphological abnormalities, enhancing their deformability and microcirculatory function. Flow cytometry using CFSE + labeling verified the higher persistence of supercooled RBCs in circulation post-transfusion, affirming their enhanced viability and functionality. These findings suggest that supercooling preservation not only extends the shelf life of RBCs but also ensures their clinical efficacy, reducing the risks associated with transfusion of older blood units and improving patient outcomes. 3.4 Clinical implications and future directions. Our engineered supercooling preservation protocol offers a transformative solution to the longstanding challenges in RBC storage and transfusion medicine. By significantly reducing hemolysis, metabolic degradation, and oxidative stress, supercooling maintains RBC integrity and functionality, thereby enhancing transfusion safety and efficacy. The scalability and compatibility of our system with existing blood banking infrastructure facilitate its potential for widespread clinical adoption. Future research should focus on further optimizing the supercooling parameters to extend RBC shelf life beyond 63 days and address the decline in recovery rates observed at extended storage durations. Additionally, investigating the integration of novel additive solutions and conducting large-scale metabolomic and proteomic analyses could provide deeper insights into the mechanisms underlying RBC preservation and identify targets for further enhancement. [ 54 ] Clinical trials are essential to validate the safety and efficacy of supercooling-preserved RBCs in human transfusions, paving the way for regulatory approval and routine clinical use. Ultimately, the adoption of supercooling preservation technology has the potential to revolutionize blood banking, ensuring a more reliable and high-quality blood supply, thereby improving healthcare delivery and patient care on a global scale. 4 Conclusions In summary, we present an engineered supercooling preservation system that extends the shelf life of large-volume (100 ml) red blood cell (RBC) suspensions in commercial PVC blood bags. By integrating paraffin oil sealing with precise thermal control at − 8°C, our protocol minimizes hemolysis, metabolic degradation, and oxidative stress, while preserving RBC membrane integrity and functionality for up to 63 days. In vivo transfusion studies using New Zealand white rabbits demonstrate that supercooling-preserved RBCs achieve superior post-transfusion recovery rates compared to conventionally stored controls, underscoring the clinical viability and enhanced transfusion quality of our method. Materials and Methods Study design and ethical approval . This study was approved by the Ethics Committee of the First Affiliated Hospital of Army Medical University, PLA (Approval Number: (A) KY2021071). All experiments were conducted in accordance with ethical standards. RBC samples were obtained from healthy donors at the Department of Transfusion Medicine, First Affiliated Hospital of Army Medical University, Chongqing, China. Approximately two units (~ 400 ml each) of packed RBCs (pRBCs) containing 100 ml of MAP additive solution were utilized for all experiments. Supercooling preservation protocol . We developed an engineered supercooling preservation system using commercial PVC blood bags. Each blood bag was securely affixed to a rigid baseplate using double-sided tape to prevent deformation during storage. For the supercooling groups, 100 ml of RBC suspension was transferred into the sealed PVC blood bag and topped with 8 ml of paraffin oil (Sigma-Aldrich, USA) to minimize ice nucleation ( Fig. S2 ). The sealed bags were then stored at -8°C in a temperature-controlled refrigerator (BD-168WGHECD; Haier, China). Control samples were stored at 4°C in a separate refrigerator (BCD-628WACW; Haier, China). Temperature stability was monitored continuously using T-type thermocouples attached to the surface of each blood bag. Optimization of supercooling conditions . To identify optimal conditions for supercooling preservation, we conducted an orthogonal experimental design considering six key factors: storage temperature, RBC volume, placement angle, cooling rate, baseplate material, and sealing oil volume ( Table 1, Fig. S1 ). Each factor was tested at three levels, resulting in 18 experimental combinations. The primary outcome was freezing frequency, defined as the ratio of frozen samples to total samples. Data were analyzed using SPSS 22.0, applying Arcsine Transformation to normalize the freezing frequency results. Detection of freezing events . Freezing events were identified through direct visual inspection and mechanical testing. Blood bags were tilted to assess flow and deformation; frozen samples remained static upon tilting, whereas supercooled samples maintained fluidity. Successful supercooling was confirmed by transparency and lucidity of the RBC suspension, indicative of the absence of ice crystals ( Fig. S7 ). Stability tests were documented in Supplementary Movie S1 . Sample preparation and storage . Two units (~ 400 ml) of RBCs (include 100 ml mannitol–adenine–phosphate storage solution (MAP): mannitol 1.457 g, glucose 0.793 g, sodium chloride 0.497 g, sodium citrate 0.15 g, sodium dihydrogen phosphate 0.094 g, citric acid 0.02 g, adenine 0.014 g) were divided into four equal segments. One segment served as the control group, stored at 4°C, while the remaining three segments were assigned to supercooling groups stored at -8°C. For each supercooling group, RBC suspensions were sealed with 8 ml of paraffin oil and stored in PVC blood bags affixed to rigid baseplates. Temperature fluctuations were minimized using different baseplate materials (copper, glass, styrofoam) to evaluate their effect on supercooling stability ( Fig. S8 ). In Vitro Analysis of RBC Preservation Hemolysis assessment . Hemolysis was quantified by centrifuging RBC samples at 3,000 rpm for 10 minutes. The supernatant was diluted with Tris-HCl (pH 8.0) and measured spectrophotometrically at 380 nm, 415 nm, and 450 nm using a microplate reader (Thermo Fisher Scientific, Finland). Free hemoglobin (F-Hb) was calculated using the formula: F-Hb(g/L)=(2OD415–OD380–OD450)×0.84. [ 55 ] Hemolysis percentage was determined as Hemolysis (%) = F-Hb × (1 – HCT) / Hb × 100. Metabolic and oxidative stress analysis . Metabolic parameters including pH, ATP, 2,3-diphosphoglycerate (2,3-DPG), glucose, and lactate were measured using specific assay kits (Beyotime, Jiang Lai, China). Reactive oxygen species (ROS) levels were evaluated with the ROS Assay Kit (S0033S; Beyotime, China), and malondialdehyde (MDA) was quantified using the lipid peroxidation MDA assay kit (S0131S; Beyotime, China). Antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were measured using respective determination kits (S0101S; Beyotime, China; A007; Jiancheng, China). All assays were performed according to manufacturer protocols. Membrane integrity and morphological analysis . Membrane damage was assessed by measuring mean cell fragility (MCF) through osmotic fragility tests. RBC suspensions were exposed to varying NaCl concentrations (0.25–0.70%) and hemolysis was quantified by measuring the optical density at 540 nm. Phosphatidylserine exposure was analyzed using Annexin V-FITC staining followed by flow cytometry (BD Biosciences, CA). Morphological changes were evaluated via blood smears observed under an inverted microscope at 600× magnification (Olympus IX53, Japan), categorizing irreversible RBCs as sphero-echinocytes and fragmented cells. In vivo transfusion studies . New Zealand white rabbits (3.0 ± 0.5 kg, male) were anesthetized with intramuscular injections of pentobarbital (30 mg/kg). Whole blood was collected aseptically into sodium citrate-containing blood bags via the carotid artery and processed to isolate RBCs by sequential centrifugation (100 g for 15 minutes to remove platelet-rich plasma, followed by 500 g for 10 minutes to remove buffy coat). Purified RBCs were resuspended in MAP solution and stored either at 4°C or using the supercooling protocol at -8°C as described above. [ 56 ] At designated storage intervals, RBCs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; abs9106; absin), washed twice in MAP, and transfused (4 ml) into the ear vein of recipient rabbits. RBC recovery was assessed at 5 minutes and 24 hours post-transfusion using flow cytometry, calculating recovery rates as (%CFSE + RBCs at 24 hours / %CFSE + RBCs at 5 minutes) × 100. [ 53 ] All animal experiments complied with the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University guidelines (Approval Number: AMUWEC20210333). Statistical analysis . Data were first assessed for normality using the Shapiro-Wilk test. Comparisons between groups were performed using Student’s two-tailed t-test with the assumption of equal variances, implemented in SPSS 22.0. Data are presented as mean ± standard deviation, with statistical significance set at p < 0.05. Specific sample sizes for each analysis are detailed in the corresponding figure legends. Declarations Ethics Approval and Consent to participate: All participants were over 18 years old and provided written informed consent for sample collection and analysis. The research procedure was approved by the Ethics Committee of the First Affiliated Hospital of Army Medical University, PLA (Approval Number: (A) KY2021071). All animal experiments complied with the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University guidelines (Approval Number: AMUWEC20210333). Consent for publication All authors have seen and approved the final version of the paper, and all are aware of the submission of the paper. Competing interests The authors declare no competing interests. Funding: The research was supported by the National Natural Science Foundation of China (No. 82470235, 21904104), the Natural Science Foundation of Chongqing, China (No. CSTC2021JCYJ-MSXMX0702), the National Key Research and Development Program of China (No. 2016YFC0101300) and the Shaanxi Sanqin Scholars Innovation Team Support Plan (No. XTR062021001). Author Contribution Q. Liu, S.C. Wang, C.Y. Yao and F. Xu conceived and designed the study. Q. Liu, S.C. Wang, C.Y. Yao and F. Xu wrote the manuscript and extensively edited the manuscript. Q. Liu and R. H. Diao collected the blood samples. Q. Liu performed experiments. Q. Liu, S.C. Wang and H.S. Huang analyzed the data. Q. Liu and J. Yan made the graphs to visualize the data. C.Y. Yao and S.C. Wang provided resources and support. 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Blood. 2021; 137(17), 2285-2298. A. Arav, Cryopreservation by Directional Freezing and Vitrification Focusing on Large Tissues and Organs. Cells. 2022; 11(7). Q. Meng et al. , Hypoxic storage of erythrocytes slows down storage lesions and prolongs shelf‐life. Journal of Cellular Physiology. 2019; 234(12), 22833-22844. K. Hagisawa et al. , Efficacy of Resuscitative Transfusion With Hemoglobin Vesicles in the Treatment of Massive Hemorrhage in Rabbits With Thrombocytopenic Coagulopathy and Its Effect on Hemostasis by Platelet Transfusion. Shock. 2018; 50(3), 324-330. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Supplementary Information Additional experimental details, including orthogonal experiment tables (Table S1), supplementary figures (Figs. S1-S8), and a demonstration movie (Movie S1), are provided in the supplementary materials. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6029466","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":418468526,"identity":"fb1e03ed-e145-4ae7-9c75-980238f8eeb9","order_by":0,"name":"Qi Liu","email":"","orcid":"","institution":"Third Military Medical University (Army Medical University)","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Liu","suffix":""},{"id":418468527,"identity":"99a0cfa2-7ad0-4f4f-b100-17aa50558f01","order_by":1,"name":"Shichun Wang","email":"","orcid":"","institution":"Third Military Medical University (Army Medical University)","correspondingAuthor":false,"prefix":"","firstName":"Shichun","middleName":"","lastName":"Wang","suffix":""},{"id":418468531,"identity":"de9b6cf4-76d5-4be1-8e27-6c6cc7dad89f","order_by":2,"name":"Jie Yan","email":"","orcid":"","institution":"Third Military Medical University (Army Medical University)","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Yan","suffix":""},{"id":418468533,"identity":"ab38e6b3-69fc-4e75-8fe5-1336c06d3291","order_by":3,"name":"Ronghua Diao","email":"","orcid":"","institution":"Third Military Medical University (Army Medical University)","correspondingAuthor":false,"prefix":"","firstName":"Ronghua","middleName":"","lastName":"Diao","suffix":""},{"id":418468534,"identity":"6c4dc7d1-8021-4c76-8799-4d4e6a1ac270","order_by":4,"name":"Haishui Huang","email":"","orcid":"","institution":"Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Haishui","middleName":"","lastName":"Huang","suffix":""},{"id":418468535,"identity":"4444a0ff-1dec-46c7-a911-cec112dadba2","order_by":5,"name":"Feng Xu","email":"","orcid":"","institution":"Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Xu","suffix":""},{"id":418468536,"identity":"98ca82ae-52bc-49fe-9515-68ffa418912f","order_by":6,"name":"Chunyan Yao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYJACZih1AMpPIFoLW2IDqVp4DInTYnD88MHPhW13Evtn93x/dDPnMAM/e44Bw88deLScSUuWntn2LHHGnbMbm3O3HWaQ7HljwNh7Bo+WAzlmzLxthxMbbuRCtBjcyDFgZmzDo+X8G4iW+TdyHoK12BPUcgNqy4YbOYwQWyQIaJG88SxZmufcYeONN9IMZ+duS+eROPOs4GAvHi1855MPfuYpOyw770byg8+526zl+NuTNz74iUeLwgEI7dgAFeABEQdwa2BgkIcqtcenaBSMglEwCkY4AACcVloNFAGUggAAAABJRU5ErkJggg==","orcid":"","institution":"Third Military Medical University (Army Medical University)","correspondingAuthor":true,"prefix":"","firstName":"Chunyan","middleName":"","lastName":"Yao","suffix":""}],"badges":[],"createdAt":"2025-02-14 09:53:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6029466/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6029466/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77302730,"identity":"02599129-87b5-4c2b-b76e-829162414154","added_by":"auto","created_at":"2025-02-27 08:42:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":320820,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptimization of supercooling preservation conditions\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003eExperimental steps for engineered supercooling preservation.\u003cstrong\u003e (B-D)\u003c/strong\u003eTemperature profiles during cooling for different baseplate materials, the temperature recorded at blood bag top, middle and bottom position with a wrap-copper \u003cstrong\u003eB)\u003c/strong\u003e, glass\u003cstrong\u003e C)\u003c/strong\u003e, styrofoam \u003cstrong\u003eD)\u003c/strong\u003e during the whole cooling process. \u003cstrong\u003e(E)\u003c/strong\u003e Effect of different thermal conductivity material as wrap-baseplate on the cooling rate analysis. \u003cstrong\u003e(F)\u003c/strong\u003e Temperature fluctuations and stability analysis for copper, glass, and styrofoam wraps. \u003cstrong\u003e(G)\u003c/strong\u003e Freezing frequency across various subzero temperatures. Freezing frequencies of 100 ml pRBCs suspensions sealed by 8 ml paraffin oil with different wrap-baseplate, stored under different temperatures (-8, -9, -10, -11, -12 and -13°C) for 2 days, per group tested once with 10 replicate samples (N=10). \u003cstrong\u003e(H-J)\u003c/strong\u003eComparative analysis of freezing frequencies among different baseplate materials. \u003cstrong\u003eH)\u003c/strong\u003e Cooling rate of wrap-styrofoam with different refrigerator temperature.\u003cstrong\u003e I) \u003c/strong\u003eThe temperature fluctuates of wrap-styrofoam with different refrigerator temperature during long-term storage. \u003cstrong\u003eJ) \u003c/strong\u003eIllustration of temperature heat transfer for supercooling preservation. slower cooling rate to the supercooling temperature and minimal temperature fluctuations during storage contribute to supercooling preservation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6029466/v1/1108966688f7e7b45dabd16c.png"},{"id":77303356,"identity":"610fd5c0-1da9-4639-86da-a75be4f3f4f5","added_by":"auto","created_at":"2025-02-27 08:50:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":279927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e analysis hemolysis and metabolic degradation of RBC\u003c/strong\u003e.\u003cstrong\u003e (A)\u003c/strong\u003e Mechanism of RBC storage lesion, including metabolic damage, oxidative damage, membrane damage, mechanical injury, solution injury, toxic injury.\u003cstrong\u003e (B)\u003c/strong\u003e Visual inspection of RBC supernatants post-centrifugation. Photos of RBC supernatant after centrifugation at 0, 14, 28, 42, 49, 56, 63, and 70 days.\u003cstrong\u003e (C) \u003c/strong\u003eMean values of hemolysis over 63 days. \u003cstrong\u003e(D-H)\u003c/strong\u003eMetabolic parameters: glucose, lactate, pH, ATP, and 2,3-DPG levels. Mean values of glucose \u003cstrong\u003eD)\u003c/strong\u003e, lactate \u003cstrong\u003eE)\u003c/strong\u003e, pH\u003cstrong\u003e F)\u003c/strong\u003e, ATP \u003cstrong\u003eG)\u003c/strong\u003e, 2,3-DPG\u003cstrong\u003e H) \u003c/strong\u003ein RBC samples at 0, 14, 28, 42, 49, 56, and 63 days. Control (blue) and supercooled (green). Data are shown as the mean ± SD from six biological replicates (n=6). independent samples t-test, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ns, nonsignificant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6029466/v1/c53923bcaff1575cd66a768e.png"},{"id":77302734,"identity":"00d35934-77e2-46d6-882e-89be70a45f16","added_by":"auto","created_at":"2025-02-27 08:42:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":381349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e analysis oxidative stress and membrane integrity of RBC.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eSchematic of storage-induced RBC morphologic change.\u003cstrong\u003e \u003c/strong\u003eHealthy discocytes into non-healthy echinocytes and sphero-echinocytes. \u003cstrong\u003e(B-E)\u003c/strong\u003e Oxidative stress markers: ROS, MDA, SOD, and CAT levels. Mean values of ROS \u003cstrong\u003eB)\u003c/strong\u003e, MDA \u003cstrong\u003eC)\u003c/strong\u003e, CAT \u003cstrong\u003eD)\u003c/strong\u003e, and SOD \u003cstrong\u003eE)\u003c/strong\u003e in RBC samples at 0, 14, 28, 42, 49, 56, and 63 days. Control (blue) and supercooled (green). Data are shown as the mean ± SD from six biological replicates (n=6). independent samples t-test, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ns, nonsignificant.\u003cstrong\u003e (F-H)\u003c/strong\u003e Membrane integrity and morphological changes: MCF, PS exposure, and RBC morphology. \u003cstrong\u003eF)\u003c/strong\u003e Mean values of Osmatic fragility test. To characterize RBC membrane integrity, we conducted the osmotic fragility test by resuspending the stored RBCs in a hypotonic solution. Mean cell fragility (MCF) reflects the NaCl concentration at which 50% hemolysis occurs. Data are shown as the mean ± SD from three biological replicates (n=3). \u003cstrong\u003eG)\u003c/strong\u003e PS exposure in the supercooled group was significantly lower than that in the control group (statistical result). Data are shown as the mean ± SD from three biological replicates (n=3). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; ns, nonsignificant. \u003cstrong\u003eH)\u003c/strong\u003e Morphological changes in the control group were mainly irreversible at the end of the preservation period. The morphological changes in the supercooled group were mainly reversible at the end of the preservation period. Red arrow: irreversible changes (sphero-echinocytes and fragmented RBCs). Scale bar = 10 µm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6029466/v1/282e8e38addeb9f0b914b3b3.png"},{"id":77303357,"identity":"fffd5412-de82-47c9-bd8e-abe1e517b86a","added_by":"auto","created_at":"2025-02-27 08:50:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":436511,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etransfusion studies demonstrating post-transfusion RBC recovery rate. (A)\u003c/strong\u003e During storage, PS expression on the RBC surface can promote RBC adhesion to endothelial cells, leading to RBC clearance by macrophages. Consequently, outer leaflet PS exposure on the RBC membrane acts as an ‘eat-me’ signal.\u003cstrong\u003e (B)\u003c/strong\u003e Overview of the experimental setup for rabbit transfusion studies. Preparation of allogeneic RBC concentrates from donor rabbits and transfusion. New Zealand white rabbits were acquired and subjected to a series of steps for blood collection, centrifugation, and purification. Once prepared, the RBCs were stored under supercooling preservation. Following specific time intervals, RBCs were marked with CFSE\u003csup\u003e+\u003c/sup\u003e and transfused back into the rabbits. \u003cstrong\u003e(C)\u003c/strong\u003e Morphological analysis of transfused RBC using blood smears.\u003cstrong\u003e \u003c/strong\u003eThe morphological changes of rabbit RBC in control group and supercooling group with storage time. Red arrow: irreversible changes (sphero-echinocytes and fragmented RBCs). Scale bar = 10 µm. \u003cstrong\u003e(E)\u003c/strong\u003e Recovery rates of RBCs stored at 4°C versus supercooling conditions over 35 days. RBCs recovery \u003cem\u003ein vivo\u003c/em\u003e after transfusion 24h of supercooling preserve and control preserve on the 0, 14, 21, 28 and 35 days. Data are shown as the mean ± SD from three biological replicates (n=3). independent samples t-test, *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; ns, nonsignificant.\u003cstrong\u003e (D)\u003c/strong\u003e Flow cytometry plots showing CFSE\u003csup\u003e+ \u003c/sup\u003eRBC labeling.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6029466/v1/4d089a44bf41e8468310ae61.png"},{"id":77303358,"identity":"fff3e22a-fca2-4aa7-9e07-1b053b042264","added_by":"auto","created_at":"2025-02-27 08:50:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":208952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Different RBC preservation method and characteristic.\u003cstrong\u003e (B)\u003c/strong\u003e Supercooling preserve storage time is longer, the volume is larger, the clinical compatibility is relatively good, higher RBC recovery, no additional cryoprotectant and no additional washing processes.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6029466/v1/0b681d5738081f19ce065827.png"},{"id":78698261,"identity":"64abb3fe-c368-4a5f-872e-4bfbae7939b8","added_by":"auto","created_at":"2025-03-17 18:01:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3286712,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6029466/v1/6886826d-2ef5-4849-b1a7-98b2ace58d62.pdf"},{"id":77302764,"identity":"b849d1ee-96f4-4261-a98f-6ba346ca04d7","added_by":"auto","created_at":"2025-02-27 08:42:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10004545,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional experimental details, including orthogonal experiment tables (\u003cstrong\u003eTable S1\u003c/strong\u003e), supplementary figures (\u003cstrong\u003eFigs. S1-S8\u003c/strong\u003e), and a demonstration movie (\u003cstrong\u003eMovie S1\u003c/strong\u003e), are provided in the supplementary materials.\u003c/p\u003e","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6029466/v1/5a2c8bc1e926ca1f4f68b7ba.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Engineered Supercooling Systems for Enhanced Long-Term Preservation of Large-Volume Red Blood Cells in Commercial Blood Bags","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eRed blood cell (RBC) transfusion is one of the most common and critical medical interventions worldwide, essential for managing conditions such as acute hemorrhage, chronic anemia, and supporting surgical procedures.\u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e The yearly donation of 120\u0026nbsp;million blood units globally, yet many regions continue to face significant disparities between blood supply and clinical demand.\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e Effective storage and preservation of RBCs are crucial to ensuring a stable and safe blood supply, directly impacting patient care and transfusion outcomes.\u003csup\u003e[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCurrently, RBC suspensions are stored at 4\u0026deg;C in polyvinylchloride (PVC) blood bags for up to 42 days.\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e However, RBC units experience \"storage lesions\" within two weeks at hypothermic conditions. These storage lesions include reduced levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG), increased oxidative stress, and membrane protein alterations, all of which compromise RBC viability and efficacy.\u003csup\u003e[\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e Clinical studies have linked the transfusion of older RBC units (\u0026gt;\u0026thinsp;2 weeks) to elevated risks of postoperative complications, infections, and higher mortality rates.\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e While cryopreservation extends RBC shelf life beyond a decade using high concentrations of glycerol (\u0026gt;\u0026thinsp;20%) as a cryoprotective agent, it introduces significant challenges.\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e The addition and removal of glycerol cause osmotic stress and hemolysis, and the deglycerolization process is laborious and time-consuming, delaying emergency transfusions.\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e Additionally, cryopreservation risks cryoinjuries such as intracellular ice formation and recrystallization, further reducing RBC viability.\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e Alternative methods like freeze-drying, though promising for room-temperature storage, still expose RBCs to ice-induced damage, resulting in reduced viability.\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRecent advancements have explored novel cryoprotectants (e.g., trehalose, tricine, nanomaterials) and innovative storage technologies (e.g., anaerobic storage, hydrogel encapsulation).\u003csup\u003e[\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e Despite promising laboratory results, these methods often involve complex procedures, severe hemolysis, small volume, low recovery rates, and lack scalability for clinical application. Supercooling has emerged as a transformative approach, maintaining RBCs below their freezing point without ice formation, typically between \u0026minus;\u0026thinsp;20\u0026deg;C and \u0026minus;\u0026thinsp;3\u0026deg;C.\u003csup\u003e[\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31 CR32\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e This method minimizes ice-mediated damage and metabolic activity, preserving cell integrity over extended periods. Previous studies have successfully supercooled small volumes (1 ml) of RBC suspensions in polystyrene rigid tubes for up to 100 days using paraffin oil sealing, achieving minimal hemolysis and high hemoglobin recovery rates.\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHowever, supercooling preservation is still limited to small-volume storage and is insufficient to meet the requirements for large-volume RBC preservation in clinical settings. Currently, maintaining adequate RBC supplies in blood banks relies on advanced storage systems utilizing commercial blood bags constructed from flexible, sterile, gas-permeable PVC materials. These commercial blood bags preserve cellular integrity through three key mechanisms: 1) minimizing mechanical hemolysis via compliant and super smooth surfaces, 2) facilitating essential gas exchange through selective membrane permeability, and 3) maintaining metabolic homeostasis and reducing oxidative stress through optimal additive solutions. This multimodal preservation approach significantly enhances transfusion safety while complying with international blood banking gold standards (AABB guidelines).\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e However, scaling supercooling preservation techniques to clinically large-volume at physiological hematocrit levels presents below technical challenges. Increased ice nucleation propensity governed by the classical nucleation theory relationship, where larger volumes and extended storage durations exponentially raise crystallization probability. Mechanical instability of PVC bags under subzero temperatures, potentially compromising bag integrity during phase change events.\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e This thermodynamic-structural paradox creates critical barriers to implementing supercooling preservation in standard blood banking operations, necessitating fundamental material science innovations coupled with advanced thermal control strategies.\u003c/p\u003e \u003cp\u003eIn this study, we present an innovative supercooling preservation protocol tailored for large-volume (100 ml) RBC suspensions stored in commercial PVC blood bags. By engineering a system that minimizes nucleation sites through paraffin oil sealing and stabilizes storage conditions with precise thermal control, we achieved successful supercooling of RBCs at -8℃ for up to 63 days under meet the Food and Drug Administration (FDA) standards condition. Compared to traditional supercooling preservation methods, we can now increase the RBC storage volume by up to 100 times, maintaining a standard hematocrit level, all while employing a standard blood bag as the storage container. Our protocol significantly reduces hemolysis, metabolic degradation, and oxidative stress while maintaining RBC membrane integrity and functionality. \u003cem\u003eIn vivo\u003c/em\u003e transfusion studies in New Zealand white rabbits demonstrated superior post-transfusion recovery rates compared to conventionally stored RBCs. This scalable and cost-effective supercooling system in commercial PVC blood bags is compatible with existing blood banking infrastructure, offering a robust and clinically relevant solution poised to transform transfusion medicine by extending RBC shelf life and enhancing transfusion outcomes.\u003c/p\u003e"},{"header":"2 Results","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Optimization of supercooling preservation conditions enhances RBC stability.\u003c/h2\u003e\n \u003cp\u003eSupercooling preservation relies on preventing heterogeneous nucleation at the liquid-air interface, typically achieved using an immiscible oil phase.\u003csup\u003e[33, 34, 38]\u003c/sup\u003e While this strategy has proven effective in rigid round-bottomed polystyrene tubes, which maintain the stability of the oil-water interface, its application in flexible PVC blood bags presents significant challenges. Flexible blood bags are prone to deformation under external forces, potentially disrupting the oil layer and initiating ice nucleation, thereby compromising the supercooled state. To address this issue, we adhered the outer surface of the PVC blood bags to a rigid baseplate using double-sided tape, thereby preventing deformation and ensuring the integrity of the oil seal during storage. This engineering modification is crucial for maintaining a stable supercooled environment in flexible blood bags.\u003c/p\u003e\n \u003cp\u003eThe maintenance of metastable supercooled states in RBC suspensions in PVC blood bags is critically influenced by multiple factors. To develop an optimized supercooling protocol for RBC in PVC blood bags, an orthogonal experimental design \u003cstrong\u003e(Table\u0026nbsp;1)\u003c/strong\u003e was implemented to systematically evaluate six critical factors at each three levels: 1) paraffin oil sealing volume, 2) storage temperature, 3) RBC suspension volume, 4) cooling rate, 5) blood bag placement angle, and 6) baseplate material. The liquid-air interface is thermodynamically conducive to heterogeneous ice nucleation due to surface tension; therefore, we minimized potential ice nucleation sites by sealing the blood bags with paraffin oil. Additionally, ice formation during the supercooling process is a stochastic phenomenon influenced by several factors, including RBC volume, storage temperature, and cooling rate. The likelihood of ice nucleation within the supercooled volume increases with larger RBC volumes, faster cooling rates, and lower storage temperatures. Furthermore, the blood bag placement angle directly modulates the RBC suspension/air interfacial area and wrap-baseplate material selection significantly impacts thermal exchange dynamics (\u003cstrong\u003eFig. S1\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eTable\u0026nbsp;1. Experimental factors with three levels.\u003c/p\u003e\n \u003ctable id=\"Tabb\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLevel\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePlacement angle\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBaseplate materials\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCooling rate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStorage volume\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStorage temperature\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSealing oil volume\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eflat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003estyrofoam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003erapid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100 ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-12\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 ml\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eII\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etilt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eglass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emiddle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150 ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-10\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 ml\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIII\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evertical\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecopper plate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eslow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200 ml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-8\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20 ml\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003eThe orthogonal experimental design systematically assessed three levels for each of the six factors, reducing the complexity from 729 potential combinations to 18 experimental setups. Each setup was evaluated based on freezing frequency, defined as the ratio of frozen samples to total samples, to determine supercooling stability. Analysis of variance revealed that storage temperature (Vk\u0026thinsp;=\u0026thinsp;0.1333) and RBC volume (Vk\u0026thinsp;=\u0026thinsp;0.1282) were the most significant factors influencing freezing frequency, followed by placement angle (Vk\u0026thinsp;=\u0026thinsp;0.0971) (\u003cstrong\u003eTable\u0026nbsp;2\u003c/strong\u003e). Cooling rate, baseplate material, and sealing oil volume also impacted freezing frequency but to a lesser extent. Under the optimal conditions identified, \u003cem\u003ei.e.\u003c/em\u003e, storing 100 ml of RBC suspensions sealed with 8 ml of paraffin oil, cooled slowly to -8.0\u0026deg;C, and maintained vertically in a monitoring refrigerator, we achieved the lowest freezing frequency, indicating enhanced supercooling stability (\u003cstrong\u003eFig. S2\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eTable\u0026nbsp;2. Orthogonal experimental design results and factor significance.\u003c/p\u003e\n \u003ctable id=\"Tabd\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTest\u003c/p\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePlacement angle\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBaseplate materials\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCooling rate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStorage volume\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStorage temperature\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSealing oil volume\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFreezing frequency (F\u003csub\u003ef\u003c/sub\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTransformed F\u003csub\u003ef\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etilt\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eglass\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003erapid\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150 ml\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-10℃\u003csub\u003e\u0026minus;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20 ml\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e80%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eflat\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecopper\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eslow\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150 ml\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-10℃\u003csub\u003e\u0026minus;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 ml\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e70%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.99\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evertical\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003estyrofoam\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003erapid\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200 ml\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-10℃\u003csub\u003e\u0026minus;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20 ml\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e70%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.99\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evertical\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003estyrofoam\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eslow\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100 ml\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-10℃\u003csub\u003e\u0026minus;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 ml\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e30%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.58\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etilt\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003estyrofoam\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emiddle\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200 ml\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-8℃\u003csub\u003e\u0026minus;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 ml\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e50%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.79\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eflat\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eglass\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003erapid\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200 ml\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-8℃\u003csub\u003e\u0026minus;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 ml\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e90%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eflat\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003estyrofoam\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emiddle\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150 ml\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-12℃\u003csub\u003e\u0026minus;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20 ml\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e80%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etilt\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003estyrofoam\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eslow\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150 ml\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-8℃\u003csub\u003e\u0026minus;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 ml\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e30%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.58\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eflat\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eglass\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eslow\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100 ml\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-8℃\u003csub\u003e\u0026minus;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20 ml\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e40%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.68\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evertical\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecopper\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emiddle\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100 ml\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-8℃\u003csub\u003e\u0026minus;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20 ml\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e20%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.46\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etilt\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecopper\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003erapid\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100 ml\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-12℃\u003csub\u003e\u0026minus;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 ml\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e70%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.99\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etilt\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eglass\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emiddle\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100 ml\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-10℃\u003csub\u003e\u0026minus;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 ml\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e50%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.79\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evertical\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eglass\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eslow\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200 ml\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-12℃\u003csub\u003e\u0026minus;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 ml\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e80%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evertical\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecopper\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003erapid\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150 ml\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-8℃\u003csub\u003e\u0026minus;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 ml\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e30%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.58\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eflat\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003estyrofoam\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003erapid\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100 ml\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-12℃\u003csub\u003e\u0026minus;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 ml\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e70%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.99\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003evertical\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eglass\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emiddle\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150 ml\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-12℃\u003csub\u003e\u0026minus;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14 ml\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e60%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.89\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eflat\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecopper\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emiddle\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200 ml\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-10℃\u003csub\u003e\u0026minus;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 ml\u003csub\u003e\u0026minus;\u0026thinsp;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e80%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etilt\u003csub\u003e\u0026minus;\u0026thinsp;II\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecopper\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eslow\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e200 ml\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-12℃\u003csub\u003e\u0026minus;I\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20 ml\u003csub\u003e\u0026minus;\u0026thinsp;III\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e70%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.99\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.91\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.49\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.08\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eII\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.83\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.57\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.49\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIII\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.61\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.93\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.34\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.34\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003ek\u003c/strong\u003e\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.1941\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0630\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0881\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.2564\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.2667\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0091\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eV\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003ek\u003c/strong\u003e\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0971\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0315\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0441\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.1282\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.1333\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0046\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cstrong\u003eNOTE\u003c/strong\u003e: I, II, and III are the summations of the transformed freezing frequencies from the tests that involve levels I, II, and III, respectively. The smallest values among I, II, and III indicate the level with the lowest freezing frequency for the sample. S\u003csub\u003ek\u003c/sub\u003e is the sum of the squares, which corresponds to the variation in the variety mean. V\u003csub\u003ek\u003c/sub\u003e is the mean square and is defined as V\u003csub\u003ek\u003c/sub\u003e = S\u003csub\u003ek\u003c/sub\u003e / f\u003csub\u003ek\u003c/sub\u003e, where f\u003csub\u003ek\u003c/sub\u003e is the number of degrees of freedom. V\u003csub\u003ek\u003c/sub\u003e represents the significance of each factor\u0026apos;s main effect, and a higher V\u003csub\u003ek\u003c/sub\u003e value signifies a greater significance for the freezing frequency. By our analysis, the best combination is slow cooling, styrofoam baseplate, 8 ml of sealing oil, -8℃, vertical orientation and 100 ml storage volume.\u003c/p\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;10 (Each condition was tested once with 10 replicate samples).\u003c/p\u003e\n \u003cp\u003eF\u003csub\u003ef\u003c/sub\u003e : Freezing frequency.\u003c/p\u003e\n \u003cp\u003eTransformed F\u003csub\u003ef\u003c/sub\u003e = arcsin(\\(\\:\\sqrt{freezing\\:frequency}\\)).\u003c/p\u003e\n \u003cp\u003eS\u003csub\u003ek\u003c/sub\u003e=(I\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;II\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;III\u003csup\u003e2\u003c/sup\u003e) / 6 - (I\u0026thinsp;+\u0026thinsp;II\u0026thinsp;+\u0026thinsp;III)\u003csup\u003e2\u003c/sup\u003e / 18; V\u003csub\u003ek\u003c/sub\u003e= S\u003csub\u003ek\u003c/sub\u003e / 2.\u003c/p\u003e\n \u003cp\u003eThe engineered steps for the supercooling preservation of RBC in commercial blood bags are illustrated in \u003cstrong\u003eFig.\u0026nbsp;1A\u003c/strong\u003e. Supercooled state stability relies on two parameters, \u003cem\u003ei.e.\u003c/em\u003e, slower cooling rate to the supercooling temperature and minimal temperature fluctuations during storage.\u003csup\u003e[28, 39\u0026ndash;44]\u003c/sup\u003e However, During the blood storage process, temperature fluctuations within the blood storage refrigerator are inevitable due to its operational mode of the cooling and heating cycles. We further analyzed the impact of various thermally conductive materials (copper, glass, styrofoam) as baseplates for supercooled storage. During cooling, we recorded temperature changes at the bottom, middle, and top of the blood bags. Copper takes about 130 mins, glass 150 mins, and styrofoam 360 mins to reach the target temperature of -8.0\u0026deg;C (\u003cstrong\u003eFig.\u0026nbsp;1B-D\u003c/strong\u003e). The refrigerator\u0026apos;s cyclical freezing compressor activity causes temperature oscillations every 30 mins, with fluctuations around 2.3\u0026deg;C. Different wraps of the blood bag modify temperature fluctuations: 2.0\u0026deg;C for wrap-copper, 0.7\u0026deg;C for wrap-glass, and 0.3\u0026deg;C for wrap-styrofoam (\u003cstrong\u003eFig.\u0026nbsp;1E-F\u003c/strong\u003e). We also assessed freezing frequencies for blood cells at various subzero storage temperatures (-8.0 to -13.0\u0026deg;C) preserved for 2 days. At -10.0\u0026deg;C, the freezing frequency is 90% for wrap-copper, 60% for wrap-glass, and 30% for wrap-styrofoam (\u003cstrong\u003eFig.\u0026nbsp;2G\u003c/strong\u003e). Specifically, the use of styrofoam as the baseplate material supported a slower cooling rate and minimized temperature fluctuations during long-term storage, further reducing the likelihood of ice nucleation (\u003cstrong\u003eFig.\u0026nbsp;1H-I\u003c/strong\u003e). Our results further confirm that maintaining a slow cooling rate and minimizing temperature fluctuations were critical in sustaining the supercooled state as evidenced by the superior performance of styrofoam-wrapped blood bags compared to those wrapped with copper or glass (\u003cstrong\u003eFig. J\u003c/strong\u003e).\u003c/p\u003e\n \u003cdiv\u003e\u003c/div\u003e\n \u003cp\u003eThese findings demonstrate that precise control of cooling parameters and sealing methods, combined with structural reinforcement of the blood bags, are essential for maintaining the supercooled state in large-volume RBC suspensions in commercial blood bags. The optimized protocol not only minimizes ice nucleation but also ensures scalability and reliability, making it suitable for clinical applications. By addressing the inherent challenges associated with flexible blood bags, our engineered supercooling preservation system significantly enhances RBC stability, laying the groundwork for improved long-term storage solutions in transfusion medicine.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Supercooling preservation minimizes hemolysis and metabolic degradation of RBC.\u003c/h2\u003e\n \u003cp\u003eTo evaluate the efficacy of supercooling preservation in maintaining RBC integrity and metabolic stability, we conducted comprehensive \u003cem\u003ein vitro\u003c/em\u003e analyses over a 63-day storage period (\u003cstrong\u003eFig.\u0026nbsp;2A, S3\u003c/strong\u003e). Hemolysis is a critical indicator of RBC preservation quality, with the FDA standard set at below 1%.\u003csup\u003e[45]\u003c/sup\u003e Hemolysis was assessed by measuring free hemoglobin (F-Hb) levels in the supernatant post-centrifugation. Visual inspection of RBC supernatants (\u003cstrong\u003eFig.\u0026nbsp;2B\u003c/strong\u003e) corroborated these findings, with supercooling-preserved samples displaying less discoloration, indicative of reduced hemolysis. The supercooling group exhibits a significantly lower hemolysis rate of 0.76% at day 63 compared to the control group\u0026rsquo;s 0.88% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (\u003cstrong\u003eFig.\u0026nbsp;2C\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eTo assess metabolic stability, we measured key parameters including glucose consumption, lactate accumulation, pH levels, adenosine triphosphate (ATP), and 2,3-diphosphoglycerate (2,3-DPG) concentrations. The supercooling group maintains significantly higher glucose levels and exhibits lower lactate accumulation compared to the control group, with notable differences at 42 days (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002 for glucose; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for lactate) (\u003cstrong\u003eFig.\u0026nbsp;2D-E\u003c/strong\u003e). Additionally, the decline in pH is less pronounced in the supercooling group, reaching 6.43 at day 63 versus 6.19 in the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (\u003cstrong\u003eFig.\u0026nbsp;2F\u003c/strong\u003e). ATP levels, essential for RBC functionality, remain significantly higher in the supercooling group (3.06 nmol/mg) compared to the control group (2.17 nmol/mg) at 42 days (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cstrong\u003e(Fig.\u0026nbsp;2G)\u003c/strong\u003e. The concentration of 2,3-DPG, crucial for oxygen release capacity, shows a markedly slower decline in the supercooling group, with both groups leveling off post 42 days (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.206) (\u003cstrong\u003eFig.\u0026nbsp;2H\u003c/strong\u003e). These results indicate that supercooling preservation effectively decelerates metabolic degradation of RBC, maintaining essential biochemical functions over extended storage durations.\u003c/p\u003e\n \u003cdiv\u003e\u003c/div\u003e\n \u003cp\u003eCollectively, these results demonstrate that supercooling preservation at -8\u0026deg;C significantly minimizes hemolysis and metabolic degradation of RBC. The maintained levels of ATP and 2,3-DPG, indicate that supercooling effectively preserves RBC functionality and viability over prolonged storage periods. Consequently, our supercooling protocol offers a superior preservation method, mitigating the hemolysis and metabolic lesions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003e2.3 Supercooling preservation reduces oxidative stress and maintains RBC membrane integrity.\u003c/h2\u003e\n \u003cp\u003eTo evaluate the impact of supercooling preservation on oxidative stress and membrane integrity in RBCs, we conducted a series of biochemical assays and morphological analyses over a 63-day storage period \u003cstrong\u003e(Fig.\u0026nbsp;3A)\u003c/strong\u003e. We measured ROS and MDA levels to assess oxidative stress, and evaluated the activities of superoxide dismutase (SOD) and catalase (CAT) to determine the RBCs\u0026apos; antioxidative defense capacity. Additionally, we examined membrane integrity through osmotic fragility tests and phosphatidylserine (PS) exposure, and analyzed RBC morphology using blood smears.\u003c/p\u003e\n \u003cp\u003eWe observed that RBCs preserved under supercooling conditions exhibit significantly lower increases in ROS and MDA levels compared to the control group stored at 4\u0026deg;C (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cstrong\u003e(Fig.\u0026nbsp;3B-C)\u003c/strong\u003e. Specifically, ROS levels in the supercooling group rise modestly over time, whereas the control group shows a higher increase, indicating higher oxidative stress in conventional storage. Similarly, MDA concentrations, a marker of lipid peroxidation, remain relatively stable in the supercooling group but increase significantly in the control group, reflecting reduced lipid membrane damage in supercooled RBCs.\u003c/p\u003e\n \u003cp\u003eFurthermore, the activities of antioxidant enzymes SOD and CAT remain largely stable in the supercooling group throughout the storage period, with only minor decreases observed towards day 63 (\u003cstrong\u003eFig.\u0026nbsp;3D-E\u003c/strong\u003e). In contrast, the control group exhibits a significant decline in CAT activity by the end of the storage period, suggesting compromised antioxidative defenses. These results indicate that supercooling preservation effectively mitigates oxidative stress, maintaining the antioxidative capacity of RBCs and protecting them from oxidative damage.\u003c/p\u003e\n \u003cp\u003eTo assess membrane integrity, we performed osmotic fragility tests and measured PS exposure on RBC surfaces. The supercooling group demonstrates lower mean cell fragility (MCF) values compared to controls, indicating enhanced membrane stability \u003cstrong\u003e(Fig.\u0026nbsp;3F)\u003c/strong\u003e. Flow cytometry analysis revealed significantly reduced PS exposure in the supercooling group at day 63 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cstrong\u003e(Fig.\u0026nbsp;3G, S4)\u003c/strong\u003e, suggesting diminished membrane asymmetry disruption and delayed recognition by phagocytes. Morphological assessments using blood smears showed that RBCs in the supercooling group retain their typical elliptical, biconcave shape with minimal formation of sphero-echinocytes and fragmented cells \u003cstrong\u003e(Fig.\u0026nbsp;3H, S5)\u003c/strong\u003e. In contrast, control RBCs exhibit pronounced morphological abnormalities, including increased echinocytosis and cell fragmentation, indicative of membrane damage and compromised structural integrity.\u003c/p\u003e\n \u003cdiv\u003e\u003c/div\u003e\n \u003cp\u003eThese findings demonstrate that supercooling preservation at -8\u0026deg;C effectively reduces oxidative stress and maintains RBC membrane integrity over extended storage periods. By minimizing ROS and MDA accumulation and preserving antioxidant enzyme activities, our supercooling protocol protects RBCs from oxidative damage. Additionally, the maintenance of membrane integrity and normal morphology in supercooled RBCs underscores the method\u0026apos;s efficacy in preserving cellular functionality and viability. Consequently, supercooling presents a superior preservation strategy by mitigating oxidative stress and membrane damage associated with conventional refrigeration, thereby enhancing the overall quality and safety of stored RBCs for transfusion.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003e2.4 Supercooling-preserved RBCs exhibit superior \u003cem\u003ein vivo\u003c/em\u003e transfusion quality.\u003c/h2\u003e\n \u003cp\u003eTo evaluate the clinical viability of supercooling-preserved RBC, we conducted \u003cem\u003ein vivo\u003c/em\u003e transfusion studies using New Zealand white rabbits. Higher percentages and longer periods of circulating RBC indicate superior RBC quality \u003cstrong\u003e(Fig.\u0026nbsp;4A)\u003c/strong\u003e, adhering to FDA guidelines, which define a successful transfusion as maintaining over 75% of transfused RBCs in circulation after 24 hours,\u003csup\u003e[3]\u003c/sup\u003e we assessed the recovery rates of supercooling-preserved RBCs compared to conventionally stored controls.\u003c/p\u003e\n \u003cp\u003eWe first isolated RBCs from rabbit whole blood by sequential centrifugation to remove platelet-rich plasma and buffy coat, followed by resuspension in mannitol\u0026ndash;adenine\u0026ndash;phosphate (MAP) solution. RBCs were then either stored at 4\u0026deg;C (control group) or subjected to our supercooling preservation protocol at \u0026minus;\u0026thinsp;8\u0026deg;C (supercooling group) for varying durations (0, 14, 21, and 35 days). At each storage interval, RBCs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE\u003csup\u003e+\u003c/sup\u003e) to facilitate tracking post-transfusion. Approximately 4 ml of labeled RBCs were transfused into recipient rabbits via the ear vein \u003cstrong\u003e(Fig.\u0026nbsp;4B)\u003c/strong\u003e. Morphological assessments using blood smears revealed that supercooled RBCs of rabbit retain their typical elliptical, biconcave shape with minimal morphological abnormalities over time \u003cstrong\u003e(Fig.\u0026nbsp;4C)\u003c/strong\u003e, whereas control RBCs exhibit increased sphero-echinocyte and fragmented cell formations indicative of membrane damage and reduced deformability. Immediately after transfusion (0-day storage), both supercooling-preserved and control RBCs demonstrate high recovery rates, with fresh RBCs achieving a 94.83% recovery at 24 hours post-transfusion. After 14 days of storage, the control group shows an 82.69% recovery rate, while the supercooling group maintains over 90% recovery. At 21 days, recovery rates declined to 65.26% in the control group and remained robust at 84.09% in the supercooling group. Although at 35 days, the supercooling group\u0026apos;s recovery rate decreases to 53.01%, falling below the FDA\u0026rsquo;s threshold, it was still significantly higher than the control group\u0026rsquo;s rate \u003cstrong\u003e(Fig.\u0026nbsp;4D)\u003c/strong\u003e. Our nonradioactive flow cytometry method, based on CFSE\u003csup\u003e+\u003c/sup\u003e labeling, demonstrated that nearly all transfused RBCs are successfully labeled, ensuring accurate tracking of RBC survival. The log fluorescence intensity versus cell counts plots confirmed consistent CFSE\u003csup\u003e+\u003c/sup\u003e staining across all samples \u003cstrong\u003e(Fig.\u0026nbsp;4E, S6)\u003c/strong\u003e.\u003c/p\u003e\n \u003cp\u003eSupercooling-preserved RBCs showed a higher persistence of CFSE\u003csup\u003e+\u003c/sup\u003e cells in circulation at 24 hours post-transfusion compared to control RBCs, corroborating the recovery rate data. These findings indicate that supercooling preservation significantly enhances the \u003cem\u003ein vivo\u003c/em\u003e transfusion quality of RBCs. Supercooled RBCs maintained higher recovery rates at critical storage intervals (14 and 21 days), demonstrating superior viability and functionality compared to conventionally stored controls. Although the recovery rate for supercooled RBCs decreased below the FDA threshold at 35 days, it remained markedly higher than that of the control group, highlighting the effectiveness of supercooling in preserving RBC quality over extended storage periods. The superior \u003cem\u003ein vivo\u003c/em\u003e recovery rates of supercooling-preserved RBCs suggest enhanced transfusion outcomes, potentially reducing the risks associated with transfusion of older blood units, such as postoperative complications and increased mortality rates. This advancement underscores the translational potential of our supercooling preservation protocol, offering a viable solution to extend RBC shelf life and improve the safety and efficacy of blood transfusions in clinical settings (\u003cstrong\u003eTable. S1\u003c/strong\u003e).\u003c/p\u003e\n \u003cdiv\u003e\u003c/div\u003e\n\u003c/div\u003e"},{"header":"3 Discussion","content":"\u003cp\u003eTraditional methods for RBC preservation, particularly cryopreservation, offer extended storage durations but are hindered by significant challenges. Cryopreservation necessitates the use of high concentrations of glycerol (\u0026gt;\u0026thinsp;20%) as cryoprotective agents (CPAs) to maintain RBC viability below \u0026minus;\u0026thinsp;65\u0026deg;C.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e However, the addition and subsequent removal of glycerol induce transient osmotic gradients, resulting in cytotoxicity and osmotic injuries that lead to severe hemolysis\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Additionally, the deglycerolization process involves multiple washing and centrifugation steps, which can delay emergency transfusions.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e Alternative preservation methods, such as freeze-drying, offer a promising solution for room-temperature storage of RBC by eliminating the need for ultra-low temperatures. However, this method is still limited by ice crystal formation during the freezing phase, which can cause mechanical damage to cellular membranes and hemoglobin structures.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e These limitations underscore the urgent need for innovative preservation techniques that can extend RBC shelf life without compromising cellular integrity or clinical utility.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Engineered supercooling protocol enhances stability and scalability for large-volume RBC preservation.\u003c/h2\u003e\n \u003cp\u003eOur study introduces an engineered supercooling preservation protocol specifically designed to address the challenges associated with large-volume RBC storage in commercial PVC blood bags. By integrating paraffin oil sealing with precise thermal control mechanisms, we successfully maintained 100 ml of RBC suspensions at \u0026minus;\u0026thinsp;8\u0026deg;C for up to 63 days (\u003cstrong\u003eFig.\u0026nbsp;1A\u003c/strong\u003e). The use of paraffin oil effectively eliminates heterogeneous nucleation at the liquid-air interface, a critical factor in preventing ice crystal formation in the metastable supercooled state.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e Additionally, adhering the flexible PVC blood bags to rigid baseplates using double-sided tape minimized deformation and ensured the stability of the oil seal during storage, a key innovation that differentiates our protocol from previous approaches.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e The orthogonal experimental design optimized six critical factors (storage temperature, RBC volume, placement angle, cooling rate, baseplate material, and sealing oil volume), resulting in enhanced supercooling stability with minimal freezing events (\u003cstrong\u003eTable. 2\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eAlthough hypothermic storage (4\u0026deg;C) and cryopreservation are widely utilized for RBC preservation, both approaches exhibit critical limitations that may compromise transfusion efficacy and safety. For hypothermic storage, the restricted shelf life (typically\u0026thinsp;\u0026le;\u0026thinsp;42 days) is accompanied by a progressive decline in RBC quality. In contrast, cryopreservation extends storage duration to years but introduces technical and biological challenges\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. One recent advance is the preservation of RBCs at -8.0\u0026deg;C for an impressive 70 days without freezing by adding a low concentration of glycerol combined with PEG-400 to reduce the freezing point. However, given the tedious rewarming and washing processes and very small storage volume (5 ml), its clinical practicality remains limited\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Our engineering strategy not only ensures the reliability and reproducibility of the supercooling state in large-volume suspensions in commercial blood bags but also highlights the scalability and practicality of our preservation system for clinical applications (\u003cstrong\u003eFig.\u0026nbsp;5\u003c/strong\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Supercooling mitigates metabolic and oxidative stress, preserving RBC functionality.\u003c/h2\u003e\n \u003cp\u003eSupercooling preservation significantly mitigates metabolic and oxidative stress of RBC, thereby maintaining cellular functionality and viability over extended storage periods. Our results demonstrate that supercooled RBCs exhibit reduced hemolysis (0.76% in supercooling vs 0.88% in controls after 63 days) and maintain higher levels of essential metabolic markers such as glucose, ATP, and 2,3-diphosphoglycerate (2,3-DPG) compared to conventionally stored RBCs. The slower glycolysis rate in supercooled RBCs leads to delayed lactate accumulation and less pronounced pH drops, indicating a deceleration of metabolic degradation.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e Oxidative stress was an important mechanism of injury at subzero temperatures. Blood bags, optimize oxygen and nutrient exchange due to gas-permeable surfaces, provide a more controlled oxygen environment. In addition, the choice of an appropriate additive solution (AS) for RBC is crucial for supercooling preservation at low temperatures.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e In this study, we selected the latest and most comprehensive formulation of mannitol-adenine-phosphate (MAP) solution as the AS for RBC storage. Compared to the other AS, the RBC stored in MAP solution experienced lower hemolysis and ensured continued glycolytic activity during supercooling preservation, primarily due to its multifaceted mechanisms in mitigating oxidative damage, stabilizing metabolic homeostasis. For example, mannitol scavenges oxygen radical via its hydroxyl groups and optimizes antioxidant capacity through the citrate-phosphate buffer system, directly attenuating oxidative stress on membrane lipids and hemoglobin\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. By maintaining metabolic homeostasis and reducing oxidative insults, supercooling preservation preserves RBC membrane integrity and prevents morphological transformations, such as echinocytosis and cell fragmentation. These biochemical and structural protections collectively enhance the overall quality and functionality of RBCs, ensuring their efficacy for transfusion purposes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Supercooling-preserved RBC demonstrate superior transfusion outcomes with clinical potential.\u003c/h2\u003e\n \u003cp\u003eThe ultimate test of RBC preservation efficacy lies in their performance during transfusion. Our \u003cem\u003ein vivo\u003c/em\u003e studies using New Zealand white rabbits demonstrate that supercooling-preserved RBCs exhibit superior post-transfusion recovery rates compared to conventionally stored controls. Following storage for 28 days, supercooled RBCs maintained recovery rates exceeding 75%, significantly outperforming the control group, which showed recovery rates of 50.13%, respectively. Although recovery rates for supercooled RBCs declined to 53.01% at 35 days, this still represented a substantial improvement over the control group (25.69%) and highlighted the potential for further optimization to meet FDA guidelines for successful transfusions.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e Morphological analyses confirmed that supercooled RBCs retained their typical elliptical, biconcave shape with minimal morphological abnormalities, enhancing their deformability and microcirculatory function. Flow cytometry using CFSE\u003csup\u003e+\u003c/sup\u003e labeling verified the higher persistence of supercooled RBCs in circulation post-transfusion, affirming their enhanced viability and functionality. These findings suggest that supercooling preservation not only extends the shelf life of RBCs but also ensures their clinical efficacy, reducing the risks associated with transfusion of older blood units and improving patient outcomes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Clinical implications and future directions.\u003c/h2\u003e\n \u003cp\u003eOur engineered supercooling preservation protocol offers a transformative solution to the longstanding challenges in RBC storage and transfusion medicine. By significantly reducing hemolysis, metabolic degradation, and oxidative stress, supercooling maintains RBC integrity and functionality, thereby enhancing transfusion safety and efficacy. The scalability and compatibility of our system with existing blood banking infrastructure facilitate its potential for widespread clinical adoption. Future research should focus on further optimizing the supercooling parameters to extend RBC shelf life beyond 63 days and address the decline in recovery rates observed at extended storage durations. Additionally, investigating the integration of novel additive solutions and conducting large-scale metabolomic and proteomic analyses could provide deeper insights into the mechanisms underlying RBC preservation and identify targets for further enhancement.\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e Clinical trials are essential to validate the safety and efficacy of supercooling-preserved RBCs in human transfusions, paving the way for regulatory approval and routine clinical use. Ultimately, the adoption of supercooling preservation technology has the potential to revolutionize blood banking, ensuring a more reliable and high-quality blood supply, thereby improving healthcare delivery and patient care on a global scale.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eIn summary, we present an engineered supercooling preservation system that extends the shelf life of large-volume (100 ml) red blood cell (RBC) suspensions in commercial PVC blood bags. By integrating paraffin oil sealing with precise thermal control at − 8°C, our protocol minimizes hemolysis, metabolic degradation, and oxidative stress, while preserving RBC membrane integrity and functionality for up to 63 days. \u003cem\u003eIn vivo\u003c/em\u003e transfusion studies using New Zealand white rabbits demonstrate that supercooling-preserved RBCs achieve superior post-transfusion recovery rates compared to conventionally stored controls, underscoring the clinical viability and enhanced transfusion quality of our method.\u003c/p\u003e "},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eStudy design and ethical approval\u003c/b\u003e. This study was approved by the Ethics Committee of the First Affiliated Hospital of Army Medical University, PLA (Approval Number: (A) KY2021071). All experiments were conducted in accordance with ethical standards. RBC samples were obtained from healthy donors at the Department of Transfusion Medicine, First Affiliated Hospital of Army Medical University, Chongqing, China. Approximately two units (~ 400 ml each) of packed RBCs (pRBCs) containing 100 ml of MAP additive solution were utilized for all experiments.\u003c/p\u003e\u003cp\u003e \u003cb\u003eSupercooling preservation protocol\u003c/b\u003e. We developed an engineered supercooling preservation system using commercial PVC blood bags. Each blood bag was securely affixed to a rigid baseplate using double-sided tape to prevent deformation during storage. For the supercooling groups, 100 ml of RBC suspension was transferred into the sealed PVC blood bag and topped with 8 ml of paraffin oil (Sigma-Aldrich, USA) to minimize ice nucleation (\u003cb\u003eFig. S2\u003c/b\u003e). The sealed bags were then stored at -8°C in a temperature-controlled refrigerator (BD-168WGHECD; Haier, China). Control samples were stored at 4°C in a separate refrigerator (BCD-628WACW; Haier, China). Temperature stability was monitored continuously using T-type thermocouples attached to the surface of each blood bag.\u003c/p\u003e\u003cp\u003e \u003cb\u003eOptimization of supercooling conditions\u003c/b\u003e. To identify optimal conditions for supercooling preservation, we conducted an orthogonal experimental design considering six key factors: storage temperature, RBC volume, placement angle, cooling rate, baseplate material, and sealing oil volume (\u003cb\u003eTable\u0026nbsp;1, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Each factor was tested at three levels, resulting in 18 experimental combinations. The primary outcome was freezing frequency, defined as the ratio of frozen samples to total samples. Data were analyzed using SPSS 22.0, applying Arcsine Transformation to normalize the freezing frequency results.\u003c/p\u003e\u003cp\u003e \u003cb\u003eDetection of freezing events\u003c/b\u003e. Freezing events were identified through direct visual inspection and mechanical testing. Blood bags were tilted to assess flow and deformation; frozen samples remained static upon tilting, whereas supercooled samples maintained fluidity. Successful supercooling was confirmed by transparency and lucidity of the RBC suspension, indicative of the absence of ice crystals (\u003cb\u003eFig. S7\u003c/b\u003e). Stability tests were documented in \u003cb\u003eSupplementary Movie S1\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e \u003cb\u003eSample preparation and storage\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eTwo units (~ 400 ml) of RBCs (include 100 ml mannitol–adenine–phosphate storage solution (MAP): mannitol 1.457 g, glucose 0.793 g, sodium chloride 0.497 g, sodium citrate 0.15 g, sodium dihydrogen phosphate 0.094 g, citric acid 0.02 g, adenine 0.014 g) were divided into four equal segments. One segment served as the control group, stored at 4°C, while the remaining three segments were assigned to supercooling groups stored at -8°C. For each supercooling group, RBC suspensions were sealed with 8 ml of paraffin oil and stored in PVC blood bags affixed to rigid baseplates. Temperature fluctuations were minimized using different baseplate materials (copper, glass, styrofoam) to evaluate their effect on supercooling stability (\u003cb\u003eFig. S8\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e \u003cb\u003eIn Vitro Analysis of RBC Preservation\u003c/b\u003e \u003c/p\u003e\u003cp\u003e \u003cb\u003eHemolysis assessment\u003c/b\u003e. Hemolysis was quantified by centrifuging RBC samples at 3,000 rpm for 10 minutes. The supernatant was diluted with Tris-HCl (pH 8.0) and measured spectrophotometrically at 380 nm, 415 nm, and 450 nm using a microplate reader (Thermo Fisher Scientific, Finland). Free hemoglobin (F-Hb) was calculated using the formula: F-Hb(g/L)=(2OD415–OD380–OD450)×0.84.\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e Hemolysis percentage was determined as Hemolysis (%) = F-Hb × (1 – HCT) / Hb × 100.\u003c/p\u003e\u003cp\u003e \u003cb\u003eMetabolic and oxidative stress analysis\u003c/b\u003e. Metabolic parameters including pH, ATP, 2,3-diphosphoglycerate (2,3-DPG), glucose, and lactate were measured using specific assay kits (Beyotime, Jiang Lai, China). Reactive oxygen species (ROS) levels were evaluated with the ROS Assay Kit (S0033S; Beyotime, China), and malondialdehyde (MDA) was quantified using the lipid peroxidation MDA assay kit (S0131S; Beyotime, China). Antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were measured using respective determination kits (S0101S; Beyotime, China; A007; Jiancheng, China). All assays were performed according to manufacturer protocols.\u003c/p\u003e\u003cp\u003e \u003cb\u003eMembrane integrity and morphological analysis\u003c/b\u003e. Membrane damage was assessed by measuring mean cell fragility (MCF) through osmotic fragility tests. RBC suspensions were exposed to varying NaCl concentrations (0.25–0.70%) and hemolysis was quantified by measuring the optical density at 540 nm. Phosphatidylserine exposure was analyzed using Annexin V-FITC staining followed by flow cytometry (BD Biosciences, CA). Morphological changes were evaluated via blood smears observed under an inverted microscope at 600× magnification (Olympus IX53, Japan), categorizing irreversible RBCs as sphero-echinocytes and fragmented cells.\u003c/p\u003e\u003cp\u003e \u003cb\u003eIn vivo transfusion studies\u003c/b\u003e. New Zealand white rabbits (3.0 ± 0.5 kg, male) were anesthetized with intramuscular injections of pentobarbital (30 mg/kg). Whole blood was collected aseptically into sodium citrate-containing blood bags via the carotid artery and processed to isolate RBCs by sequential centrifugation (100 g for 15 minutes to remove platelet-rich plasma, followed by 500 g for 10 minutes to remove buffy coat). Purified RBCs were resuspended in MAP solution and stored either at 4°C or using the supercooling protocol at -8°C as described above.\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAt designated storage intervals, RBCs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; abs9106; absin), washed twice in MAP, and transfused (4 ml) into the ear vein of recipient rabbits. RBC recovery was assessed at 5 minutes and 24 hours post-transfusion using flow cytometry, calculating recovery rates as (%CFSE\u003csup\u003e+\u003c/sup\u003e RBCs at 24 hours / %CFSE\u003csup\u003e+\u003c/sup\u003e RBCs at 5 minutes) × 100.\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e All animal experiments complied with the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University guidelines (Approval Number: AMUWEC20210333).\u003c/p\u003e\u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e. Data were first assessed for normality using the Shapiro-Wilk test. Comparisons between groups were performed using Student’s two-tailed t-test with the assumption of equal variances, implemented in SPSS 22.0. Data are presented as mean ± standard deviation, with statistical significance set at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. Specific sample sizes for each analysis are detailed in the corresponding figure legends.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics Approval and Consent to participate:\u003c/h2\u003e\n\u003cp\u003eAll participants were over 18 years old and provided written informed consent for sample collection and analysis. The research procedure was approved by the Ethics Committee of the First Affiliated Hospital of Army Medical University, PLA (Approval Number: (A) KY2021071). All animal experiments complied with the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University guidelines (Approval Number: AMUWEC20210333).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have seen and approved the final version of the paper, and all are aware of the submission of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eThe research was supported by the National Natural Science Foundation of China (No. 82470235, 21904104), the Natural Science Foundation of Chongqing, China (No. CSTC2021JCYJ-MSXMX0702), the National Key Research and Development Program of China (No. 2016YFC0101300) and the Shaanxi Sanqin Scholars Innovation Team Support Plan (No. XTR062021001).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eQ. Liu, S.C. Wang, C.Y. Yao and F. Xu conceived and designed the study. Q. Liu, S.C. Wang, C.Y. Yao and F. Xu wrote the manuscript and extensively edited the manuscript. Q. Liu and R. H. Diao collected the blood samples. Q. Liu performed experiments. Q. Liu, S.C. Wang and H.S. Huang analyzed the data. Q. Liu and J. Yan made the graphs to visualize the data. C.Y. Yao and S.C. Wang provided resources and support.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe thank Shangsheng Feng, and Pengpeng Jia of Xi\u0026rsquo;an Jiaotong University for input, discussions and heat transfer technical assistance.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and Materials:\u003c/h2\u003e\n\u003cp\u003eAll data supporting the findings of this study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eM. A. 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Shock. 2018; 50(3), 324-330.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Supercooling Preservation, Red Blood Cells, Blood Bags, Metabolic Stability, Oxidative Stress","lastPublishedDoi":"10.21203/rs.3.rs-6029466/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6029466/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExtending the shelf life of red blood cells (RBCs) is vital for transfusion medicine, yet traditional storage methods like refrigeration and cryopreservation suffer from limitations such as storage lesions and ice-induced damage. We developed an improved supercooling preservation system for large-volume (100 ml) RBC suspensions in commercial polyvinylchloride (PVC) blood bags by minimizing favorable sites of ice nucleation and maintaining precise thermal control at \u0026minus;\u0026thinsp;8\u0026deg;C. This engineered protocol significantly reduces hemolysis, metabolic degradation, and oxidative stress while preserving RBC membrane integrity and functionality for up to 63 days. \u003cem\u003eIn vivo\u003c/em\u003e transfusion studies in New Zealand white rabbits demonstrate that supercooling-preserved RBCs achieve higher post-transfusion recovery rates, outperforming conventional storage methods. Our scalable and cost-effective supercooling system integrates seamlessly with existing blood banking infrastructure, addressing critical needs for extended RBC storage and improved transfusion outcomes. This advancement enhances blood supply reliability and patient care, representing a significant breakthrough in transfusion medicine.\u003c/p\u003e","manuscriptTitle":"Engineered Supercooling Systems for Enhanced Long-Term Preservation of Large-Volume Red Blood Cells in Commercial Blood Bags","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-27 08:42:40","doi":"10.21203/rs.3.rs-6029466/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b7fbd2e1-2b0a-4405-a8a1-29c698ba1eda","owner":[],"postedDate":"February 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-17T17:53:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-27 08:42:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6029466","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6029466","identity":"rs-6029466","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}