Supercooling preservation method capable of preserving functional integrity of red blood cells for long time

Abstract

The invention relates to the technical field of red blood cell preservation, and discloses a supercooling preservation method capable of preserving functional integrity of red blood cells for a long time, which comprises the following steps: step 1, fixing a PVC blood bag on a hard substrate; 2, storing 100-200ml of red blood cells in the PVC blood bag, and sealing the top of the liquid level of the red blood cells with an incompatible oil phase; 3, the PVA blood bag in the step 2 is subjected to supercooling storage. According to the invention, the PVC blood bag is combined with the paraffin oil for sealing, so that the crystallization freezing point is reduced, the storage condition is stabilized, and 100ml of red blood cells are successfully preserved at the temperature of-8 DEG C for 63 days. Tests on the preserved erythrocytes find that the method of the scheme significantly reduces hemolysis, metabolism and oxidative stress, and maintains the integrity and functionality of erythrocyte membranes. The method is expected to obviously promote the progress of red blood cell preservation technology and standard and improve the medical level of blood transfusion.

Description

Technical Field

[0001] The present invention relates to the technical field of red blood cell preservation, and in particular to a supercooling preservation method capable of preserving the functional integrity of red blood cells for a long period of time. Background Art

[0002] Red blood cell transfusion is one of the most common procedures in hospitals and is the cornerstone of many life-saving medical interventions. According to the World Health Organization (WHO), there were 118.5 million blood donations recorded worldwide in 2022, a figure that highlights the importance of blood supply on a global scale. However, despite the large number of blood donations, there is still a significant gap between supply and demand in many regions. In order to bridge this gap and enhance the effectiveness of blood transfusions, it is crucial to develop advanced red blood cell storage methods. Traditional 4°C refrigerated storage is limited by storage damage, while frozen storage is subject to the risk of ice crystal damage and cryoprotectant removal. Currently, the commonly used technology for red blood cell storage is to store packed red blood cells (pRBCs) in polyvinyl chloride (PVC) blood bags, which can be stored at 4°C for up to 35 to 42 days. However, studies have shown that even under optimal storage conditions, red blood cells will begin to suffer from "storage damage" after two weeks. These damages include changes in cell membranes, accumulation of metabolites, and changes in the intracellular environment, which together lead to a decline in red blood cell function. Transfusion of aged RBC units older than two weeks has been associated with an increased risk of postoperative complications and higher mortality.

[0003] In response to the above technical problems, scholars in the field have provided a solution by optimizing cryopreservation, which can extend the storage life of red blood cells for more than ten years. This method uses high-concentration glycerol (>20%) to store red blood cells at conditions below -65°C, but involves a cumbersome deglycerolization process and a series of risks of freezing damage. During the cryopreservation process, the addition and removal of glycerol constitute a major clinical obstacle. High-concentration glycerol may cause severe osmotic shock and severe hemolysis during the addition and removal process. In addition, the deglycerol process is cumbersome and time-consuming, involving multiple centrifugation and washing steps, which significantly delays the time for urgent blood transfusion. Cryoinjury, such as ice formation and recrystallization damage during thawing, further complicates clinical applications. Although freeze-drying has been proposed as an alternative method that can be stored at room temperature, it still exposes red blood cells to ice damage during freezing and drying, often resulting in cell damage and reduced viability.

[0004] In the pursuit of evolving transition solutions, researchers have worked hard to improve red blood cell storage methods to ensure long-term and clinical efficacy. New cryoprotectants (such as trehalose, triglycine, nanomaterials, antifreeze polymeric proteins) and technologies (anaerobic storage, hydrogel or microfiber embedding) have promoted the development of red blood cell cryopreservation, but these methods remain challenging due to complex operations, severe hemolysis, and insufficient red blood cell recovery, limiting their promotion and use in clinical practice. Therefore, it is urgent to develop a new red blood cell storage method to reduce the metabolic activity and oxidative damage of red blood cells and extend the storage period of red blood cells. Summary of the invention

[0005] The present invention aims to provide a supercooling storage method that can preserve the functional integrity of red blood cells for a long time, so as to reduce the metabolic activity and oxidative damage of red blood cells and extend the storage period of red blood cells.

[0006] To achieve the above object, the present invention adopts the following technical solution: a supercooling storage method capable of preserving the functional integrity of red blood cells for a long time, comprising the following steps:

[0007] Step 1: Fix the PVC blood bag on a hard substrate;

[0008] Step 2: 100-200 ml of red blood cells are stored in a PVC blood bag, and the top of the red blood cell liquid surface is sealed with an incompatible oil phase;

[0009] Step 3: Supercool the PVA blood bag from step 2.

[0010] Preferably, as an improvement, in step one, the hard substrate is a heat-insulating substrate.

[0011] Preferably, as an improvement, in step one, the thermal insulation substrate is a packaging polystyrene foam board.

[0012] Preferably, as an improvement, in step 2, the incompatible oil phase is paraffin oil, and the amount of paraffin oil added is 8-20% of the volume of red blood cells.

[0013] Preferably, as an improvement, in step three, the supercooled storage condition is a temperature of -12 to -8°C, and a temperature fluctuation of <0.5°C.

[0014] Preferably, as an improvement, in step three, the cooling rate is 0.022°C / min.

[0015] Preferably, as an improvement, in step one, the PVC blood bag is a commercial PVC blood bag, and the PVC blood bag is wrapped with a thermal insulation layer.

[0016] Preferably, as an improvement, the material of the thermal insulation layer is polystyrene foam.

[0017] Preferably, as an improvement, in step three, the supercooled storage conditions are: red blood cell volume 100 ml, paraffin oil addition amount is 8% of the red blood cell volume, supercooled storage temperature is -8°C, temperature fluctuation is <0.5°C, cooling rate is 0.022°C / min, and the PVC blood bag is placed vertically.

[0018] Preferably, as an improvement, the shelf life is at least 63 days.

[0019] The principle and advantages of this solution are as follows: in practical application, in the early stage of research and development, the inventors found that the supercooling storage method (below the freezing point of biological samples (between -20°C and -3°C)) can significantly reduce ice-mediated damage. Previous studies have shown that 1 ml of red blood cell suspension can be supercooled for 100 days at -16°C using 0.5 ml paraffin oil surface sealing, with almost no hemolysis and about 94% hemoglobin recovery. However, a major challenge of supercooling is how to prevent ice formation in the metastable state (supercooled state) between liquid and solid phase transitions. It is particularly difficult to achieve supercooling in large-volume red blood cell suspensions because: the larger the red blood cell storage volume, the more nucleation sites there are, and the easier it is to freeze; moreover, the larger the volume, the higher the requirements for the external environment of storage, because slight vibrations or temperature fluctuations in the outside world can easily cause the entire system to freeze quickly; and the paraffin oil surface sealing method is feasible only in the case of rigid storage containers, because rigid containers help maintain the stability of the oil-water interface. However, considering the convenience and feasibility of later clinical applications, this solution explored a long-term and stable storage method for large volumes of red blood cells based on commercial PVC blood bags (flexible blood bags are commonly used for red blood cell suspensions). However, during the technical research and development stage, flexible blood bags are prone to deformation when subjected to external forces. To solve this problem, this technical solution adheres the outer surface of the blood bag to a hard substrate to prevent deformation, and combines an immiscible oil phase to prevent heterogeneous nucleation at the liquid / air interface.

[0020] In addition, the inventors found that the stability of the supercooled state has a key influence on the effect of red blood cell storage, and the stability of the supercooled state depends on two parameters, namely slow cooling to the supercooled temperature and the minimum temperature fluctuation during storage. In order to enhance the effect of supercooled storage, this technical solution analyzes the influence of various thermal conductive materials (copper, glass and polystyrene foam) as substrates for supercooled storage, and finds that the polystyrene foam board takes 360 minutes to reach the target temperature of -8.0°C, which meets the requirements of slow cooling. In addition, by arranging thermal insulation materials (polystyrene foam) outside the PVC blood bag, it can be ensured that the temperature fluctuation during storage is less than 0.5°C. In this way, maintaining a slow cooling rate combined with smaller temperature fluctuations can ensure the stability of the supercooled state and avoid freezing of the entire system due to the external environment and temperature fluctuations.

[0021] In summary, this technical solution successfully preserved 100 ml of red blood cells at -8°C for 63 days by using PVC blood bags combined with paraffin oil seals to reduce the crystallization freezing point and stabilize storage conditions. Tests on stored red blood cells showed that the method of this solution significantly reduced hemolysis, metabolic and oxidative stress, and maintained red blood cell membrane integrity and functionality. This method is expected to improve the level of transfusion medicine and significantly promote the advancement of patient care and clinical outcomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Graph showing factors influencing the stability of the supercooled state in an embodiment of the present invention.

[0023] Figure 2 Graph showing the relationship between different substrate materials and cooling rates in an embodiment of the present invention.

[0024] Figure 3 This is the effect of different packaging materials on temperature fluctuation in the embodiments of the present invention.

[0025] Figure 4 This is the temperature variation curve of the top, middle and bottom of the blood bag in which copper is used as the outer packaging material in the embodiment of the present invention.

[0026] Figure 5 This is the temperature variation curve of the top, middle and bottom of the blood bag in which glass is used as the outer packaging material in the embodiment of the present invention.

[0027] Figure 6 This is the temperature variation curve of the top, middle and bottom of the blood bag with polystyrene foam as the outer packaging material in the embodiment of the present invention.

[0028] Figure 7 Graph showing the relationship between storage target temperature and cooling rate of different packaging materials in an embodiment of the present invention.

[0029] Figure 8 Graph showing the relationship between target storage temperature and temperature fluctuation of different packaging materials in an embodiment of the present invention.

[0030] Fig. 9 Graph showing the relationship between different supercooled storage temperatures and red blood cell freezing frequencies in an embodiment of the present invention.

[0031] Fig.10 The present invention is a flowchart of a supercold storage method for preserving the functional integrity of red blood cells for a long time according to an embodiment of the present invention.

[0032] Fig.11 Schematic diagram of oxidative damage during blood storage in an embodiment of the present invention.

[0033] Fig.12 Schematic diagram of the supernatant of blood centrifugation after storage in an embodiment of the present invention.

[0034] Fig.13 This is a graph showing the effect of supercold storage on free hemoglobin in an example of the present invention.

[0035] Fig.14 This is a comparison chart of the hemolysis rate between the supercold storage and the control group in the embodiment of the present invention.

[0036] Fig.15 Flow chart of the metabolic process of red blood cells in an embodiment of the present invention.

[0037] Fig.16 Graph showing the effect of supercooling storage on glucose content in the examples of the present invention.

[0038] Fig.17 This is the effect of supercooling storage on lactic acid content in the examples of the present invention.

[0039] Fig.18 This is the effect of supercooling storage on pH in the examples of the present invention.

[0040] Fig.19 This is the effect of supercooling storage on ATP levels in the examples of the present invention.

[0041] Fig. 20 This is the effect of supercold storage on 2,3-DPG levels in the examples of the present invention.

[0042] Fig.21 This is the effect of supercold storage on ROS levels in the examples of the present invention.

[0043] Fig. 22 This is the effect of supercold storage on MDA levels in the examples of the present invention.

[0044] Fig.23 This is the effect of supercold storage on CAT levels in the examples of the present invention.

[0045] Fig.24 This is the effect of supercold storage on SOD levels in the examples of the present invention.

[0046] Fig.25 Graph showing the morphological changes of red blood cells during supercold storage in an embodiment of the present invention.

[0047] Fig.26 This is the effect of supercooling storage on the MCF value in the examples of the present invention.

[0048] Fig. 27 This is the effect of supercooling storage on PS exposure in the examples of the present invention.

[0049] Fig.28 This is the effect of supercold storage on red blood cell PS damage in the examples of the present invention.

[0050] Fig.29This is the effect of supercold storage on the morphology of red blood cells in the examples of the present invention.

[0051] Fig.30 Graph showing the percentage of irreversible red blood cells in the supercooling group and the control group in an embodiment of the present invention.

[0052] Fig.31 Schematic diagram of phagocytes clearing red blood cells from blood circulation in an embodiment of the present invention.

[0053] Fig.32 This is a flow chart for preparing allogeneic red blood cell concentrate from donor rabbits and performing transfusion in an embodiment of the present invention.

[0054] Fig.33 This is a comparison chart of the recovery of supercooled stored red blood cells and control stored red blood cells at different storage times in the embodiment of the present invention.

[0055] Fig.34 This is a graph showing the logarithmic fluorescence intensity versus cell number results in an embodiment of the present invention.

[0056] Fig.35 This is a fluorescence image of red blood cells labeled with CFSE+ in vitro in an example of the present invention.

[0057] Fig.36 This is a graph showing the in vivo recovery rate results 24 hours after blood transfusion in rabbits in the examples of the present invention. DETAILED DESCRIPTION

[0058] The following is further described in detail through specific implementations, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following implementations are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.

[0059] like Fig.10 A supercooling storage method for preserving the functional integrity of red blood cells for a long time comprises the following steps:

[0060] Step 1: fix the commercial PVC blood bag on a hard substrate, which is a heat-insulating substrate, and in this solution, a packaging polystyrene foam board is preferred;

[0061] Step 2: 100 ml of red blood cells are placed in a PVC blood bag, and the top of the liquid surface of the red blood cells is sealed with paraffin oil. The amount of paraffin oil added is 8 to 20 ml, and in this embodiment, it is 8 ml;

[0062] Step 3: Store the PVC blood bag containing the red blood cell suspension at -8°C with a cooling rate of 0.022°C / min.

[0063] The red blood cell storage method of the present technical solution significantly reduces hemolysis, metabolic and oxidative stress, and maintains the integrity and functionality of the red blood cell membrane.

[0064] Experimental Example 1 Determination of the Optimal Conditions for Supercold Storage of Red Blood Cells

[0065] In order to develop a supercold storage protocol, we conducted an orthogonal experiment to determine the optimal conditions for maintaining red blood cell suspension in PVC blood bags. Six potential factors affecting supercold storage were identified using orthogonal testing, including storage temperature, volume, placement angle, cooling rate, base material, and sealing oil volume. Each factor had three different levels, as shown in Table 1. An orthogonal table was created using SPSS22.0. According to the experimental design, the freezing frequency (freezing frequency = number of frozen samples / total number of samples) was used as the observation. In this table, we used arcsine transformation to convert the freezing frequency.

[0066] The significance of each factor was assessed based on freezing frequency, which was defined as the ratio of the number of frozen specimens to the total number of specimens. Storage temperature (Vk = 0.1333) and volume (Vk = 0.1282) were found to significantly affect freezing frequency, and the placement angle was the next significant factor (Vk = 0.0971). The optimal combination of supercold storage conditions included storing 100 ml of red blood cell suspension in a PVC blood bag, sealing with 8 ml of paraffin oil, slowly cooling to -8.0°C, and placing it vertically in the refrigerator.

[0067] Table 1 Orthogonal test results

[0068]

[0069] Note: I, II, and III are the sum of the freezing frequency transformations for the tests involving levels I, II, and III, respectively. The smallest value among I, II, and III indicates the level with the lowest freezing frequency of the samples. Sk is the sum of squares, corresponding to the change in the mean of the variable. Vk is the mean square, defined as Vk=Sk / fk, where fk is the number of degrees of freedom. Vk represents the significance of the main effect of each factor, and higher Vk values ​​indicate a higher significance of the freezing frequency. According to our analysis, the best combination is slow cooling, foam board, 8 ml sealing oil, -8°C, vertical orientation, and 100 ml storage capacity. N=10 (10 replicate samples were tested under each condition).

[0070] Experimental Example 2 Effect of Cooling Rate and Temperature Fluctuation on Supercooling Storage

[0071] The stability of the supercooled state depends on two parameters, namely, slow cooling to the supercooled temperature and minimal temperature fluctuations during storage ( Figure 1 ).

[0072] Cooling rate: To analyze the effect of enhancing supercold storage, the influence of different thermally conductive materials (copper, glass, and polystyrene foam) as the substrate for supercold storage was analyzed. During the cooling process, the temperature changes at the bottom of the blood bag were recorded. The results are shown in Figure 2. Figure 2 As shown: It takes about 130 minutes for copper as a substrate to cool from room temperature to -8°C, 150 minutes for glass, and 360 minutes for polystyrene foam board to reach the target temperature of -8.0°C.

[0073] Temperature fluctuation: It is critical to maintain a stable temperature during supercooled storage. The cyclic freezing compressor activity of the refrigerator causes a temperature fluctuation of about 2.3°C every 30 minutes. During the cooling process, the effects of different materials (copper, glass, and polystyrene foam) as the outer cover of the PVC blood bag on the temperature fluctuation were investigated. The temperature changes at the top, middle, and bottom of the blood bag during the cooling process were also recorded. The results are as follows: Figure 3-6 As shown: Different blood bag packaging modifies the temperature fluctuation: 2.0°C for copper packaging, 0.7°C for glass packaging, and 0.3°C for polystyrene foam packaging ( Figure 3 ). And combined Figure 4-6 It can be seen that the temperature at the bottom of the PVC blood bag is the highest and the top is the lowest, indicating that cooling is mainly carried out from the top to the bottom. As the storage target temperature decreases, the cooling rate increases slightly ( Figure 7 ) and keep the temperature fluctuation very small (<0.5℃) during supercooling storage, especially for polystyrene foam packaging ( Figure 8 ).

[0074] Experimental Example 3: Study on the relationship between supercooled storage temperature and freezing frequency of blood cells

[0075] The freezing frequency of blood cells stored for 2 days in different packaging materials (copper, glass, polystyrene foam) and different cryogenic storage temperatures (-8.0 to -13.0°C) was evaluated. Fig. 9 As shown: At -10.0°C, the freezing frequency was 90% for copper packaging, 60% for glass packaging, and 30% for polystyrene foam packaging. These results indicate that slow cooling rates and minimizing temperature fluctuations are essential for successful supercold storage of red blood cells at -8.0°C, and polystyrene foam packaging is more conducive to supercold storage.

[0076] Experimental Example 4: In vitro analysis of storage damage of red blood cells

[0077] 1. Effect of supercold storage on hemolysis rate

[0078] Storage of blood can lead to a series of pathological reactions, collectively referred to as "storage damage". Common types of oxidative damage include Fig.11 Hemolysis is essential for the preservation of red blood cells and is permitted at levels below 1% in the United States and below 0.8% in Europe.

[0079] In order to evaluate the effect of long-term storage, the stored blood was centrifuged and the supernatant ( Fig.12 ), the free hemoglobin (F-Hb) level was measured to evaluate cell hemolysis, and a control (stored at 4°C) was set up at the same time.

[0080] The test method for F-Hb level is as follows: the suspended red blood cell sample is centrifuged at 3000rpm for 10min. 30μl of supernatant is collected, added to a 96-well plate, evenly mixed with 270μl Tris-HCl (pH 8.0), and the optical density (OD) is measured at 380nm, 415nm and 450nm wavelengths using a microplate reader (Thermo Fisher Scientific, Finland). Hemoglobin (Hb) and hematocrit (HCT) are measured using a blood cell counter (XS-900i; Sysmex, Japan). The calculation formulas for F-Hb and hemolysis rate are as follows:

[0081] F-Hb (g / L)=(2OD415-OD380-OD450)×0.84;

[0082] Hemolysis rate (%) = F-Hb × (1 – HCT) / Hb × 100

[0083] The results showed that compared with the control group, the F-Hb level in the supercold storage group increased less over time ( Fig.13 After 63 days, the hemolysis rate of the control group was 0.88%, while that of the supercold storage group was 0.76% ( Fig.14 ). These results indicate that storage of RBCs at supercooled conditions of -8.0°C significantly reduced hemolysis and prolonged shelf life.

[0084] 2. Effects of supercold storage on metabolism and oxidative damage in red blood cells

[0085] During the 63-day storage period, glucose, lactate, pH, ATP, and 2,3-DPG levels were analyzed in the control (4°C constant temperature storage) and supercooled storage groups to study energy metabolism ( Fig.15 ). The test methods are as follows:

[0086] ① Glucose detection:

[0087] a. Take 5 μl of standard or sample into a PCR tube.

[0088] b. Add 185 μl of Glucose Assay Reagent to make the final volume 190 μl.

[0089] c. After vortex mixing, centrifuge at 5000×g for a few seconds to allow the liquid to settle to the bottom of the tube.

[0090] d. Heat at 95°C for 8 minutes on a PCR instrument and then cool to 4°C.

[0091] e. After cooling to 4°C, remove the PCR tubes. Aspirate 180 μl of liquid from each tube into a clean 96-well plate. Be careful to avoid bubbles when transferring liquid.

[0092] f. Measure absorbance at 630nm.

[0093] g. Calculate the glucose concentration in the sample based on the standard curve.

[0094] ② Lactic acid detection: Take 20 μl of standard or sample, add lactic acid detection reagent, mix well, react immediately at 37°C in the dark for 30 minutes, and read the absorbance at 450 nm;

[0095] ③ pH value: Take 4 ml of the test sample and put it into a 15 ml EP tube, and measure it directly using a pH instrument (PB-21; Sartorius, Germany).

[0096] ④ATP detection: Add 100 microliters of ATP detection working solution to the detection well. Leave it at room temperature for 3-5 minutes to consume all the background ATP, thereby reducing the background. b. Add 20 microliters of sample or standard to the detection well or detection tube, quickly mix with a gun (micropipette), and after at least 2 seconds, use a chemiluminometer to measure the RLU value.

[0097] ⑤2,3-DPG detection: Detection is carried out using the Elisa method. The specific steps are as follows:

[0098] 1. Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes;

[0099] 2. Set up standard wells and sample wells, and add 50 μL of standards of different concentrations to each standard well;

[0100] 3. First add 10μL of the sample to be tested to the sample well, then add 40μL of sample diluent; do not add to the blank well.

[0101] 4. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard well and sample well, seal the reaction wells with a sealing film, and incubate in a 37°C water bath or incubator for 60 min.

[0102] 5. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this process 5 times (you can also use a plate washer to wash the plate).

[0103] 6. Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0104] 7. Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

[0105] The results showed that the control group consumed glucose faster than the supercooled group. Within 42 days, the glucose level in the control group decreased from 31.55mmol / L to 13.35mmol / L, while the supercooled group showed a gradual decrease throughout the storage period. At the end of the storage period, the glucose level in the supercooled group was significantly higher (p = 0.002; Fig.16 ).

[0106] In addition, the lactic acid accumulation in the control group was significant at 42 days, reaching 16.36mmol / L, while that in the supercooled storage group was 14.81mmol / L (p<0.05; Fig.17 ).

[0107] Although lactate levels in both groups gradually increased after 42 days, the pH drop in the supercold storage group was milder than that in the control group. By day 63, the pH in the supercold storage group dropped to 6.43, while the pH in the control group was recorded as 6.19 (p < 0.05; Fig.18 ).

[0108] The ATP concentration of the control group was 2.17 nmol / mg at 42 days, while that of the supercold storage group reached 3.06 nmol / mg. At the end of the storage period, the supercold storage group still maintained a relatively high ATP level (p<0.05; Fig.19 ).

[0109] The 2,3-DPG level in the control group decreased sharply in the first 28 days, but the rate of decrease was significantly slower in the supercold storage group, and both groups stabilized after 42 days (p = 0.206; Fig. 20 ). Overall, these findings suggest that the metabolism of RBCs stored at -8.0°C is significantly slowed compared with RBCs stored at 4°C.

[0110] The production of ROS (Reactive Oxygen Species) increased in both groups during storage, but the increase in the supercooled storage group was significantly smaller (p<0.05; Fig.21 The MDA level in the control group increased sharply during storage, while the MDA (malondialdehyde) level in the supercooled storage group was relatively stable (p<0.05; Fig. 22 ). CAT and SOD levels in the supercold storage group remained statistically similar to those in the control group during most of the storage period ( Fig.23 ,24). However, at the end, the control group showed a significant decrease in intracellular CAT concentrations compared with the supercryopreservation group (p<0.05). These results emphasize that supercryopreservation can reduce oxidative damage in stored erythrocytes.

[0111] 3. Effects of supercold storage on membrane damage and morphology of red blood cells

[0112] Experimental method: Prepare blood cell smears and observe cell morphology under a microscope; stain red blood cell membranes with flow cytometry antibodies and detect using a flow cytometer.

[0113] Storage of RBCs under traditional conditions often compromises the integrity of the RBC membrane, leading to the formation of exocytic granules and hemolysis ( Fig.25 ). A higher mean cell fragility (MCF) value indicates poor red blood cell membrane quality.

[0114] During the 63-day storage period, the MCF value of the supercooled storage group increased slightly ( Fig.26 ), indicating that supercold storage can prevent the degradation of erythrocyte membrane quality. We further measured the amount of phosphatidylserine (PS) exposure to explore the effect of supercold storage on membrane structure. The control group showed a significant increase in PS exposure level on day 63 ( Fig. 27 ,28). The supercold storage group still maintained a mostly oval, uniform, and smooth red blood cell morphology after 63 days, while the control group showed significant cell damage and irregularity. Irreversible red blood cells included spherical-spiny cells and fragmented red blood cells. The proportion of irreversible red blood cells increased in both groups during storage, but the increase in the supercold storage group was smaller than that in the control group ( Fig.29 ,30). These results are very important because they show that supercold storage can reduce membrane damage and morphological changes in supercooled erythrocytes.

[0115] 4. Effect of supercold storage on the quality of concentrated red blood cell transfusion

[0116] To evaluate the quality of supercold stored RBCs, the recovery time of RBCs in vivo after transfusion was assessed. A higher percentage of circulating RBCs and a longer circulation time indicated superior RBC quality ( Fig.31 According to FDA guidelines, successful transfusion is defined as more than 75% of the transfused red blood cells remaining in circulation after 24 hours. Based on this concept, we evaluated the efficacy of transfusion of supercooled concentrated blood in rabbits ( Fig.32 We first examined the morphology of rabbit RBCs over time and found that supercold storage also better maintained the morphology of rabbit RBCs ( Fig.33). At the same time, the technical solution also adopts a non-radioactive method for measuring red blood cell survival based on CFSE+RBCs flow cytometry (the specific experimental method is: by collecting rabbit whole blood and preparing red blood cell suspension by centrifugation, then CFSE+ labeled red blood cells in vitro, the labeled red blood cells are returned to the rabbit, and the rabbit whole blood is collected 5 minutes and 24 hours after the return, and the CFSE+ labeled red blood cell content is detected by flow cytometry, and the 24-hour red blood cell survival rate = (CFSE+ labeled red blood cell content at 24 hours / CFSE+ labeled red blood cell content at 5 minutes) × 100%), considering that the labeling has no corruption or obvious elution advantage. Displayed in the format of logarithmic fluorescence intensity versus cell number, almost all rabbit-red blood cells are stained with CFSE+ fluorescent dye ( Fig.34 ,35). Using this approach, we evaluated the persistence of circulating red blood cells after transfusion in rabbits ( Fig.36 ). Initially, almost 94.83% of the 0-day (fresh) red blood cells were still circulating 24 hours after transfusion. After 14 days of storage, 82.69% of the red blood cells in the control group were still circulating 24 hours after transfusion, while 92.01% in the supercold storage group. After 21 days of storage, 65.26% of the red blood cells in the control group were still circulating 24 hours after transfusion, while 84.09% in the supercold storage group. After 35 days of storage, 53.01% of the red blood cells in the supercold storage group (less than 75%) were still circulating 24 hours after transfusion, while 25.69% in the control group.

[0117] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A supercold storage method for preserving the functional integrity of red blood cells for a long time, characterized in that: The steps include: Step 1: Fix the PVC blood bag on a hard substrate; Step 2: 100-200 ml of red blood cells are stored in a PVC blood bag, and the top of the red blood cell liquid surface is sealed with an incompatible oil phase; Step 3: Supercool the PVA blood bag from step 2.

2. A supercold storage method for preserving the functional integrity of red blood cells for a long time according to claim 1, characterized in that: In step 1, the hard substrate is a heat-insulating substrate.

3. A supercold storage method for preserving the functional integrity of red blood cells for a long time according to claim 2, characterized in that: In step 1, the heat insulating substrate is a packaging polystyrene foam board.

4. A supercold storage method for preserving the functional integrity of red blood cells for a long time according to claim 3, characterized in that: In step 2, the incompatible oil phase is paraffin oil, and the amount of paraffin oil added is 8-20% of the volume of red blood cells.

5. A supercold storage method for preserving the functional integrity of red blood cells for a long time according to claim 4, characterized in that: In step 3, the supercooled storage condition is a temperature of -12 to -8°C, and a temperature fluctuation of <0.5°C.

6. A supercold storage method for preserving the functional integrity of red blood cells for a long time according to claim 5, characterized in that: In step 3, the cooling rate is 0.022°C / min.

7. A supercold storage method for preserving the functional integrity of red blood cells for a long time according to claim 6, characterized in that: In step 1, the PVC blood bag is commercial PVC, and the PVC blood bag is wrapped with a thermal insulation layer.

8. A supercold storage method for preserving the functional integrity of red blood cells for a long time according to claim 7, characterized in that: The material of the thermal insulation layer is polystyrene foam.

9. A supercold storage method for preserving the functional integrity of red blood cells for a long time according to claim 8, characterized in that: In step 3, the supercooled storage conditions are: the red blood cell volume is 100 ml, the amount of paraffin oil added is 8% of the red blood cell volume, the supercooled storage temperature is -8°C, the temperature fluctuation is <0.5°C, the cooling rate is 0.022°C / min, and the PVC blood bag is placed vertically.

10. A supercold storage method for preserving the functional integrity of red blood cells for a long time according to claim 8, characterized in that: In step 3, the retention period is at least 63 days.

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