Application of circadian rhythm regulation-based macrophage MHC-II molecular expression in heart transplantation

Abstract

The invention discloses application of macrophage MHC-II molecular expression based on circadian rhythm regulation in heart transplantation, and belongs to the technical field of biological medicine. According to the method, the optimal obtaining opportunity of the donor heart is obtained through circadian rhythm synchronization, circadian rhythm molecular diagnosis and algorithm fitting and optimization, then when the donor heart is obtained, an anti-MHC-II neutralizing antibody is poured, meanwhile, the anti-MHC-II neutralizing antibody is added into a soaking preservation solution, the anti-MHC-II neutralizing antibody directly acts on the immune system of the donor, and the anti-MHC-II neutralizing antibody directly acts on the immune system of the donor. The expression level of MHC-II molecules on the surface of the macrophage is reduced. According to the method, the defects and deficiencies existing in an existing heart transplantation technology are overcome, and particularly the problems that the donor circadian rhythm is not included in a transplantation distribution system and the donor heart obtaining time cannot be effectively optimized are solved. According to the method, immunological rejection after heart transplantation can be effectively reduced, the dosage and side effects of an immunosuppressant are reduced, and meanwhile the transplantation success rate and the long-term survival rate of a patient are increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of macrophage MHC-II molecule expression regulated by circadian rhythm in heart transplantation. Background Art

[0002] Heart transplantation is an effective means for treating end-stage heart diseases, but the shortage of donor hearts and the high rejection rate after transplantation limit its wide application. A variety of donor- and recipient-related risk factors jointly affect the long-term prognosis of heart transplantation. Therefore, discovering and exploring new donor risk factors are expected to effectively improve the survival rate of heart transplantation.

[0003] Existing heart transplantation techniques face many challenges in donor selection and the transplantation process, and the donor circadian rhythm, as a potential prognostic predictor, has not been fully considered. The current transplantation allocation system is mainly based on basic matching parameters of donors and recipients, such as blood type, body weight, heart size, etc., but does not take the donor circadian rhythm into account. In addition, the time of donor heart acquisition has a significant impact on the prognosis after transplantation, but existing technical means cannot effectively achieve the optimal timing of donor heart acquisition. In recent years, studies have found that the circadian rhythm plays an important regulatory role in the function of immune cells. This finding suggests that the donor circadian rhythm may affect the prognosis of heart transplantation. In the transplantation immune response, the donor's professional antigen-presenting cells, especially macrophages, are one of the key factors leading to transplantation rejection. Macrophages present antigens through MHC-II molecules on their surface, activate T cells, and thus trigger an immune response. That is, the activated or inhibited state of the donor circadian rhythm may affect the immune response and rejection risk after transplantation by influencing the activity and function of immune cells. However, there is currently no systematic treatment strategy based on regulating macrophage MHC-II molecule expression by circadian rhythm applied to the field of heart transplantation. Therefore, there is an urgent need for a method to optimize macrophage MHC-II molecule expression by regulating the donor circadian rhythm, thereby reducing the immune rejection reaction after heart transplantation. Summary of the Invention

[0004] The present invention aims to solve the defects and deficiencies existing in the existing heart transplantation techniques, especially the problem that the donor circadian rhythm is not included in the transplantation allocation system and the time of donor heart acquisition cannot be effectively optimized. The present invention optimizes macrophage MHC-II molecule expression by regulating the donor circadian rhythm, can effectively reduce the immune rejection reaction after heart transplantation, reduce the dosage and side effects of immunosuppressants, and at the same time improve the transplantation success rate and the long-term survival rate of patients.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides an application of a product for regulating MHC-II molecule expression in the preparation of products for heart preservation, heart preconditioning, and / or heart transplantation, and the application is carried out by obtaining a donor heart at 5-7 in the morning and down-regulating the expression level of MHC-II molecules therein.

[0007] Preferably, the method for obtaining the time of obtaining the donor heart includes the following steps: First, within 48 hours to 24 hours before obtaining the donor heart, place the donor in a cycle of 12 hours of light and 12 hours of darkness to optimize the expression of the donor's circadian rhythm genes by simulating the natural circadian rhythm; then obtain the optimal obtaining time through algorithm fitting.

[0008] More preferably, the light intensity is 300-500 lux.

[0009] Still more preferably, the algorithm is based on the Cosinor model.

[0010] Even more preferably, the method further includes the step of detecting the expression level of circadian rhythm factors.

[0011] Preferably, the method for down-regulating the expression level of MHC-II molecules includes the following steps: First, at the time of obtaining the donor heart, perfuse an anti-MHC-II neutralizing antibody, and then add an anti-MHC-II neutralizing antibody to the preservation solution.

[0012] More preferably, the perfusion dose of the anti-MHC-II neutralizing antibody is 1-10 mg / kg body weight, the perfusion flow rate is 10-30 ml / min, and the perfusion time is 5-10 minutes.

[0013] Still more preferably, the perfusion dose of the anti-MHC-II neutralizing antibody is 5 mg / kg body weight, the perfusion flow rate is 20 ml / min, and the perfusion time is 8 minutes.

[0014] More preferably, the concentration of the anti-MHC-II neutralizing antibody in the preservation solution is 1-10 mg / L, the temperature of the preservation solution is 2-6 °C, and the preservation time is 0.5-2 hours.

[0015] Still more preferably, the concentration of the anti-MHC-II neutralizing antibody in the preservation solution is 5 mg / L, the temperature of the preservation solution is 4 °C, and the preservation time is 1 hour.

[0016] In view of the defects and deficiencies in the existing heart transplantation technology, such as the shortage of donor hearts, the high rejection rate after transplantation, and the inability to effectively optimize the time of obtaining donor hearts, the present invention proposes a new technical solution. Through improvements such as circadian rhythm optimization, HLA-II (MHC-II molecule) matching typing, and anti-MHC-II neutralizing antibody treatment, the present invention can bring the following beneficial effects:

[0017] 1. Improve the survival rate after transplantation

[0018] By obtaining the donor heart at the optimal time and combining with HLA-II (MHC-II molecule) matching and typing, the present invention can significantly improve the survival rate after transplantation. Experimental data show that the 3-year survival rate of donor hearts obtained in the morning reaches 84.4%, while that of donor hearts obtained in the afternoon / evening is 59.3%. In addition, by subgrouping according to the expression level of the antagonistic molecule NR1D2 of the core circadian rhythm molecule ARNTL, it is found that the 3-year survival rate of donor hearts obtained in the morning and with high expression of NR1D2 is as high as 93.8%, while that of donor hearts obtained in the afternoon / evening and with low expression of NR1D2 is only 48.0%. These data indicate that the technical solution of the present invention can significantly improve the survival rate after transplantation.

[0019] 2. Reduce immune rejection

[0020] The present invention perfuses anti-MHC-II neutralizing antibody during the acquisition of the donor heart and adds anti-MHC-II neutralizing antibody to the preservation solution, which directly acts on the immune system of the donor and reduces the expression level of MHC-II molecules on the surface of macrophages. Experimental data show that for donor hearts treated with anti-MHC-II neutralizing antibody, the degree of immune rejection after transplantation is milder and the dosage of immunosuppressants is reduced. This improvement can effectively reduce immune rejection after transplantation and improve the transplantation success rate.

[0021] 3. Reduce the dosage and side effects of immunosuppressants

[0022] Since the technical solution of the present invention can significantly reduce immune rejection after transplantation, the dosage of immunosuppressants can be reduced. This can not only reduce the economic burden on patients, but also reduce the side effects brought by immunosuppressants, such as increased risk of infection and tumors. Experimental data show that for donor hearts treated with the technical solution of the present invention, the dosage of immunosuppressants after transplantation is reduced and the quality of life of patients is significantly improved.

[0023] 4. Simple operation and easy for clinical application

[0024] The technical solution of the present invention has a simple operation and is easy for clinical application. By perfusing anti-MHC-II neutralizing antibody during the acquisition of the donor heart and adding anti-MHC-II neutralizing antibody to the preservation solution, the immune state of the donor heart can be optimized without complex surgical operations or additional equipment. This improvement makes the technical solution of the present invention easy to be popularized and applied in clinics. Description of the Drawings

[0025] Figure 1 It is the circadian rhythm of the risk value of the 5-year survival rate after transplantation and different donor heart acquisition times in Example 1 of the present invention;

[0026] Figure 2 This is the result of the impact of obtaining the best timing combined with molecular diagnosis on the survival rate after transplantation in Example 1 of the present invention;

[0027] Figure 3 This is the result of the impact of obtaining the best timing combined with HLA-II (MHC-II molecule) matching typing on the survival rate after transplantation in Example 1 of the present invention;

[0028] Figure 4 This is the result of the impact of knocking out the core circadian molecule Arntl in mice on the survival of grafts in Example 1 of the present invention;

[0029] Figure 5 This is the change in the proportion of macrophages (APCs) highly expressing MHC-II at different circadian stages in mice in Example 1 of the present invention;

[0030] Figure 6 This is the reversal of the survival difference caused by circadian rhythm by treatment with anti-MHC-II neutralizing antibody in Example 1 of the present invention. Detailed implementation manners

[0031] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention. The reagents, reagent kits and instruments used in the following examples can all be obtained commercially. The methods used in the examples are the same as the commonly used methods unless otherwise specified.

[0032] The technical solutions of the present invention will be further elaborated in detail below in conjunction with the examples.

[0033] Example 1

[0034] This example provides a method for regulating the expression of macrophage MHC-II molecules based on the donor's circadian rhythm , the steps are as follows:

[0035] 1. Optimization of the acquisition time of the donor heart

[0036] The acquisition time of the donor heart has a significant impact on the prognosis after transplantation, and the existing technical means cannot effectively achieve the best timing for obtaining the donor heart. The present invention proposes the following technical solutions to optimize the acquisition time of the donor heart:

[0037] (1) Circadian rhythm synchronization

[0038] Within 48 to 24 hours before the donor heart is harvested, the donor is placed in a cycle of 12 hours of light and 12 hours of darkness, with a light intensity of 300 - 500 lux (300 lux is used in the following specific experiments), using the visible light band, and any light device within approximately 400 to 700 nanometers can be used. By simulating the natural circadian rhythm, the expression of the donor's clock genes is optimized. This light regulation method can ensure that the donor's circadian rhythm is in the best state.

[0039] (2) Circadian rhythm molecular diagnosis

[0040] At 12 hours, 8 hours, and 4 hours before the donor heart is harvested, circadian rhythm-related factors such as melatonin and cortisol are detected through serum. The levels of these biomarkers can reflect the circadian rhythm state of the donor, thereby providing a basis for determining the optimal harvesting time. For example, the melatonin level reaches its peak at night and drops to its lowest in the early morning. By detecting the melatonin level, the trough period of the donor's circadian rhythm can be determined, and it may be more beneficial to harvest the donor heart at this time.

[0041] (3) Algorithm fitting to obtain the optimal harvesting time

[0042] Based on cosine analysis (Cosinor) analysis (period = 24) to fit the donor's circadian rhythm phase, and the donor heart is harvested at the optimal harvesting time (close to 6 am and with the lowest melatonin expression level). At this time, the expression level of the donor macrophage HLA-DR (corresponding to MHC-II in mice) molecule is the lowest. This algorithm-based optimization method can more accurately determine the optimal harvesting time of the donor heart, thereby improving the transplantation success rate and the long-term survival rate of patients.

[0043] 2. The circadian rhythm core molecule BMAL1 regulates the expression of MHC-II molecules

[0044] Donor macrophages present antigens through MHC-II molecules, activate T cells, and thus trigger an immune response. For the donor heart harvested at the optimal harvesting time using the above method, the present invention proposes the following technical solutions to regulate the expression of macrophage MHC-II molecules:

[0045] When the donor heart is harvested, by perfusing anti-MHC-II neutralizing antibodies and adding anti-MHC-II neutralizing antibodies to the immersion preservation solution at the same time, it directly acts on the donor's immune system and reduces the expression level of MHC-II molecules on the surface of macrophages.

[0046] Among them, the antibody product information:

[0047] Mouse model: For the mouse model, neutralizing antibodies specific for mouse MHC-II molecules (I-A) can be used. Relevant information can be found in InVivoMAb anti-mouse MHC class II (I-A) (BioXCell Cat#BE0178). This antibody can react with multiple mouse MHC Class II haplotypes and has been reported to inhibit I-A-restricted T cell responses.

[0048] Human donors: For human donors, neutralizing antibodies specific for human MHC-II molecules (HLA-DR) can be used. Relevant information can be found in InVivoMAb anti-human / monkey MHC class II (HLA-DR) (BioXCell Cat#BE0306). This antibody can be used to block MHC class II HLA-DR in vitro and is suitable for models of humans and monkeys.

[0049] The specific steps are as follows:

[0050] a. Perfusion of anti-MHC-II neutralizing antibody:

[0051] When harvesting the donor heart, the anti-MHC-II neutralizing antibody (dose: 1 - 10 mg / kg body weight, 5 mg / kg body weight was used in the following specific experiment) is directly perfused into the donor heart through a heart perfusion system. The flow rate of the perfusion fluid is controlled at 10 - 30 ml / min (20 ml / min was used in the following specific experiment) to ensure uniform distribution of the antibody in the heart tissue. The perfusion time is 5 - 10 minutes (8 minutes was used in the following specific experiment) to ensure that the antibody fully acts on macrophages in the heart tissue.

[0052] b. Addition of anti-MHC-II neutralizing antibody to the preservation solution:

[0053] After harvesting the donor heart, the heart is immersed in UW preservation solution containing anti-MHC-II neutralizing antibody ( CSS, 1L-China, LOT: CHD072424). The concentration of anti-MHC-II neutralizing antibody in the preservation solution is 1 - 10 mg / L (5 mg / L was used in the following specific experiment). The temperature of the preservation solution is controlled at 2 - 6°C (4°C in the following specific experiment) to slow down metabolic activities and extend the preservation time of the donor heart.

[0054] The preservation time is 0.5 - 2 hours (1 hour in the following specific experiment) to ensure that the antibody continuously acts on macrophages during preservation and further reduces the expression level of MHC-II molecules.

[0055] The specific experimental process of this example is as follows :

[0056] 1. Experimental design

[0057] To verify the effectiveness of the technical solutions for optimizing the donor heart acquisition time and regulating the expression of MHC-II molecules proposed in the present invention, the following experiments were designed, and corresponding control groups and experimental groups were set up.

[0058] 2. Design of experimental group and control group

[0059] Control group

[0060] Donor treatment: The donor hearts were harvested at the best time (6:00 am, corresponding to Zeitgeber Time, ZT12 after synchronization of the mouse circadian rhythm) and the worst time (6:00 pm, corresponding to ZT0 after synchronization of the mouse circadian rhythm).

[0061] Experimental group

[0062] Donor treatment: The donor hearts were harvested at the best time (6:00 am, corresponding to Zeitgeber Time, ZT12 after synchronization of the mouse circadian rhythm) and the worst time (6:00 pm, corresponding to ZT0 after synchronization of the mouse circadian rhythm), and anti-MHC-II neutralizing antibody was perfused at the time of harvest (dose: 5 mg / kg body weight), and anti-MHC-II neutralizing antibody was added to the preservation solution (concentration: 5 mg / L).

[0063] 3. Experimental methods and results

[0064] (1) Through multi-center retrospective studies, the circadian rhythm of the risk value of five-year survival rate after transplantation was determined for different donor heart acquisition times. The results are as Figure 1 shown.

[0065] The results showed that the trough period of the risk value of five-year survival rate after transplantation for different donor heart acquisition times was 06:00 (the best acquisition time period was 12 AM - 12 PM, AM group), while the peak was 18:00 (the non-best acquisition time period was 12 PM - 12 AM, PM group).

[0066] (2) Donor hearts were harvested at the best and non-best times, and molecular diagnosis (detection of circadian rhythm-related factors) was combined for analysis. The results are as Figure 2 shown.

[0067] In the study of optimizing the acquisition of donor hearts according to the circadian rhythm, we compared the effects of acquiring donor hearts in the AM and PM on the survival rate after transplantation. The results showed that the 3-year survival rate after transplantation of donor hearts acquired in the morning was significantly higher than that of donor hearts acquired in the afternoon / evening, being 84.4% and 59.3% respectively (log-rank P = 0.045). This result indicates that the time of donor heart acquisition has a significant impact on long-term survival after transplantation. Further, we detected the expression level of the antagonist molecule NR1D2 (REV-ERBα) of the donor circadian rhythm core molecule ARNTL (BMAL1) by RNA-sequencing (BGI: DNBseq TM ) and divided the donors into a high NR1D2 expression group (NR1D2 high) and a low NR1D2 expression group (NR1D2 low). Among the donors acquired in the morning, the 3-year survival rate of the NR1D2 high group was as high as 93.8%, while among the donors acquired in the afternoon / evening, the 3-year survival rate of the NR1D2 low group was only 48.0% (log-rank P = 0.029). This difference indicates that the analysis combining the circadian rhythm molecular expression level can more accurately predict the mortality rate after transplantation.

[0068] (3) In a multi-center human database, subgroup analysis was performed on the matching degree of donor-recipient HLA-DR (MHC-II molecule) genotypes by combining the histocompatibility lab test provided by UNOS (United Network for Organ Sharing) (completed by the PCR-SSP (polymerase chain reaction-sequence specific primer) method) to explore the combined effects of different donor heart acquisition time periods (morning, AM; afternoon / evening, PM) and HLA matching. The specific grouping is as follows:

[0069] AM low: Donor heart is acquired in the morning with low HLA-DR matching degree.

[0070] AM high: Donor heart is acquired in the morning with high HLA-DR matching degree.

[0071] PM low: Donor heart is acquired in the afternoon / evening with low HLA-DR matching degree.

[0072] PM high: Donor heart is acquired in the afternoon / evening with high HLA-DR matching degree.

[0073] Through long-term follow-up of the above four groups, the risk of all-cause death 5 years after transplantation was evaluated. The results are as Figure 3 shown.

[0074] Results showed that: among the groups that received donor hearts in the morning (AM low and AM high), there was no significant difference in the 5-year all-cause death risk between the group with low HLA-DR matching (AM low) and the group with high HLA-DR matching (AM high) (P>0.05), indicating that HLA matching has relatively limited impact on post-transplant survival rate during the optimal procurement time period (AM). However, among the groups that received donor hearts in the afternoon / evening (PM high), compared with the group that received donor hearts in the morning with low HLA-DR matching (AM low), the 5-year all-cause death risk was significantly increased by 1.2 times (P<0.05). This result suggests that at non-optimal procurement time period (PM), high HLA-DR matching cannot completely offset the increased death risk caused by time factors. Further analysis showed that when donor hearts were procured in the morning, HLA-DR matching could significantly reduce the increased death risk caused by non-optimal procurement time. This indicates that optimizing the donor heart procurement time and HLA-DR matching typing in combination with the circadian rhythm can significantly improve transplant prognosis. That is, procurement at the optimal time combined with HLA-DR (MHC-II molecule) matching typing significantly improves post-transplant survival rate.

[0075] (4) In a mouse model, the core circadian molecule Arntl (BMAL1) was knocked out by gene knockout technology to explore the difference in graft survival between transplanting donor hearts procured at the optimal time (mouse ZT12) and non-optimal time (mouse ZT0). Mouse source: kindly provided by Dr. Ying Xu, Soochow University, Jiangsu, China. The results are as Figure 4 shown.

[0076] Results showed that: for mice transplanted at the optimal time (ZT12), the survival time of their grafts was significantly longer than that of mice transplanted at the non-optimal time (ZT0). After transplantation at the optimal time (ZT12), the median survival time of grafts in mice with Arntl knocked out was significantly prolonged. That is, knocking out the core circadian molecule Arntl in mice delays rejection and prolongs graft survival.

[0077] (5) In this study, single-cell transcriptome sequencing technology (single-cell RNA-sequencing, 10×Genomics Chromium Next GEM Single Cell 5′ Reagent Kits v2.0, Cat#1000165) was used to perform circadian rhythm synchronization model analysis on wild-type and Arntl knockout mice. In these models, we used Seurat v5 software ( https: / / satijalab.org / seurat / )Single-cell transcriptome analysis was performed to detect the expression levels of MHC-II molecules in macrophages at different circadian phases (daytime, night). By this method, we were able to precisely evaluate how the circadian rhythm affects the expression of MHC-II molecules in macrophages and further understand its potential impact on the immune response after heart transplantation. The results are as Figure 5 shown.

[0078] The results showed that the proportion of the APC+ (high MHC-II expression) macrophage subset exhibited significant circadian rhythmic changes at different times of the day. In particular, at ZT0 (non-optimal time, equivalent to midnight in humans), the proportion of APC+ macrophages reached its peak. This finding indicates that donor hearts obtained at non-optimal times may have a higher risk of death due to an increased number of macrophages with high MHC-II expression. The high expression of MHC-II molecules may lead to stronger antigen presentation ability, thereby activating T cells and triggering a stronger immune response. This may be a key factor contributing to increased transplant rejection and reduced post-transplant survival rate.

[0079] (6) Donor hearts obtained at different circadian rhythm phases (optimal and non-optimal times) were treated with anti-MHC-II neutralizing antibody (perfusion and preservation solution treatment) and soaked for 1 hour. The orthotopic heart transplantation surgery was performed using an abdominal heart transplantation model. During the transplantation process, we paid particular attention to the preservation and treatment conditions of the donor heart to ensure that the antibody could effectively play its role. In addition, to evaluate the effect of antibody treatment, we regularly monitored the heart function after transplantation and evaluated the degree of immune rejection through histological examination. The results are as Figure 6 shown.

[0080] The results showed that treatment with anti-MHC-II neutralizing antibody could significantly reverse the effect of circadian rhythm on post-transplant survival rate (P>0.05).

[0081] Conclusion :

[0082] From the above experimental data, it can be found that the technical solution of the present invention shows significant advantages in optimizing the donor heart acquisition time and regulating the expression of MHC-II molecules in macrophages. Acquisition at the optimal time combined with molecular diagnosis and treatment with anti-MHC-II neutralizing antibody can significantly improve the post-transplant survival rate and reduce the incidence of immune rejection. These results provide strong experimental evidence for the technical solution of the present invention, demonstrating its application potential in the field of heart transplantation.

[0083] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a product for regulating the expression of MHC-II molecules in the preparation of products for cardiac preservation, cardiac pretreatment and / or cardiac transplantation, characterized in that: The application is performed by obtaining the donor heart between 5 and 7 am and down-regulating the expression level of MHC-II molecules therein.

2. The use according to claim 1, characterized in that: The method for obtaining the donor heart acquisition time comprises the following steps: first, within 48 hours to 24 hours before the donor heart is obtained, the donor is placed in a cycle of 12 hours of light and 12 hours of darkness, and the donor's biological clock gene expression is optimized by simulating the natural circadian rhythm; and then the optimal acquisition time is obtained by algorithm fitting.

3. The use according to claim 2, characterized in that: The light intensity is 300-500 lux.

4. The use according to claim 3, characterized in that: The algorithm is based on the Cosinor model.

5. The use according to claim 4, characterized in that: The method further comprises the step of detecting the expression level of the circadian rhythm factor.

6. The use according to claim 1, characterized in that: The method for down-regulating the expression level of MHC-II molecules comprises the following steps: before obtaining the donor heart, perfusing the anti-MHC-II neutralizing antibody, and then adding the anti-MHC-II neutralizing antibody to the preservation solution.

7. The use according to claim 6, characterized in that: The perfusion dose of the anti-MHC-II neutralizing antibody is 1-10 mg / kg body weight, the perfusion flow rate is 10-30 ml / min, and the perfusion time is 5-10 minutes.

8. The use according to claim 7, characterized in that: The perfusion dose of the anti-MHC-II neutralizing antibody was 5 mg / kg body weight, the perfusion flow rate was 20 ml / min, and the perfusion time was 8 minutes.

9. The use according to claim 6, characterized in that: The concentration of the anti-MHC-II neutralizing antibody in the storage solution is 1-10 mg / L, the temperature of the storage solution is 2-6° C., and the storage time is 0.5-2 hours.

10. The use according to claim 9, characterized in that: The concentration of the anti-MHC-II neutralizing antibody in the storage solution is 5 mg / L, the temperature of the storage solution is 4° C., and the storage time is 1 hour.

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