A negative hydrogen ion synergistic nerve repair stem cell composition and a preparation method thereof

Abstract

The application discloses a kind of negative hydrogen ion synergistic nerve repair stem cell composition and preparation method thereof, it is related to biological medicine technical field.A kind of negative hydrogen ion synergistic nerve repair stem cell composition, by mass parts, consisting of the following ingredients:stem cell 5-10 parts, negative hydrogen ion donor 0.05-0.1 part, neurotrophic factor mixture 0.1-1 part, cell adhesion promoter 0.02-0.05 part, antioxidant repair agent 0.05-0.2 part, base culture medium 80-90 parts.The application constructs high-efficiency antioxidant system by introducing the synergistic effect of negative hydrogen ion donor and novel antioxidant repair agent.This system can effectively remove active oxygen in the damage site, significantly reduce the damage of oxidative stress response to cells, thereby significantly improve the survival rate and antioxidant stability of stem cells in the in-vivo microenvironment, provide more stable cell basis for nerve repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a stem cell composition for neural repair with synergistic effects of negative hydrogen ions and its preparation method. Background Technology

[0002] Nerve injury and neurodegenerative diseases have become a major global health challenge, and the core of their treatment lies in repairing and restoring the function of damaged nerve tissue. Traditional treatments, such as drug intervention and surgical correction, can only relieve symptoms and cannot fundamentally repair damaged nerve cells and neural networks, thus limiting their clinical efficacy.

[0003] Stem cell therapy offers a new direction for nerve repair due to the self-renewal, multi-directional differentiation potential, and paracrine function of stem cells. Among them, bone marrow mesenchymal stem cells, adipose tissue mesenchymal stem cells, and umbilical cord mesenchymal stem cells have become research hotspots in the field of nerve repair due to their wide availability and low immunogenicity. However, the application of stem cells alone faces several bottlenecks: oxidative stress in the in vivo microenvironment leads to low stem cell survival rates and insufficient differentiation efficiency; the lack of targeted nutritional support and adhesion conditions makes it difficult for stem cells to engraft at the damaged site and exert a repairing effect. In existing nerve repair-related compositions, neurotrophic factors are mostly used alone, failing to synergistically regulate nerve cell proliferation, differentiation, and survival; the types of antioxidants are limited, and their antioxidant efficiency is insufficient, making it difficult to effectively remove excessive reactive oxygen species at the damaged site, thus failing to create a stable environment for stem cell survival and nerve repair; simultaneously, the lack of reasonable use of synergistic components such as negative hydrogen ion donors fails to fully utilize their synergistic effects of antioxidation and cell metabolism regulation, resulting in poor overall repair effects.

[0004] Furthermore, issues such as the purity of stem cell culture, the stability of component mixing, and aseptic control during the preparation of the composition directly affect the safety and efficacy of the final product. Therefore, developing a stem cell composition for nerve repair that can synergistically improve stem cell survival efficiency, enhance antioxidant capacity, provide comprehensive nutritional support, and improve adhesion conditions, thereby addressing the limitations of single-component effects and insufficient synergistic effects in existing technologies, has become an urgent need in the field of nerve repair. Summary of the Invention

[0005] The purpose of this invention is to address the problems existing in current neural repair technologies, such as low stem cell survival rate, insufficient antioxidant capacity, limited nutritional support, and lack of synergistic effects, by providing a stem cell composition for neural repair with synergistic enhancement of negative hydrogen ions and its preparation method. This composition integrates negative hydrogen ion donors, a mixture of neurotrophic factors, and antioxidant repair agents to enhance the colonization efficiency, differentiation potential, and antioxidant stability of stem cells in the damaged microenvironment, thereby achieving efficient and safe neural repair.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A stem cell composition for nerve repair with synergistic effect of negative hydrogen ions, comprising the following components by weight: 5-10 parts stem cells, 0.05-0.1 parts negative hydrogen ion donor, 0.1-1 parts neurotrophic factor mixture, 0.02-0.05 parts cell adhesion promoter, 0.05-0.2 parts antioxidant repair agent, and 80-90 parts basal culture medium;

[0008] The antioxidant repair agent is a compound shown in Formula 1:

[0009]

[0010] In Formula 1, R1 is at least one of methyl, ethyl, methoxy, and nitro.

[0011] Furthermore, the stem cells are bone marrow mesenchymal stem cells.

[0012] Furthermore, the negative hydrogen ion donor is at least one of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate.

[0013] Furthermore, the neurotrophic factor mixture is composed of brain-derived neurotrophic factor, nerve growth factor and neurotrophin-3, with a mass ratio of 2:1:1.

[0014] Furthermore, the basal culture medium is DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin antibiotics.

[0015] Furthermore, the antioxidant repair agent is any one of the following compounds:

[0016] .

[0017] Furthermore, the cell adhesion promoter is laminin.

[0018] Furthermore, the aforementioned stem cell composition for neural repair with synergistic effects of negative hydrogen ions can be used for the breeding of superior livestock breeds.

[0019] A method for preparing a stem cell composition for nerve repair with synergistic effects of negative hydrogen ions includes the following steps:

[0020] S1: Stem Cell Acquisition and Culture: The stem cells were collected and cultured in the basal culture medium at 37°C and 5% CO2 in an incubator to the 3rd-5th generation. After digestion and collection, the stem cells were washed 2-3 times with PBS buffer, centrifuged, washed again, and resuspended in the basal culture medium to adjust the stem cell concentration to 1×10⁻⁶. 6 -5×10 6cells / mL, to prepare a stem cell suspension;

[0021] S2: Preparation of substrate solution: Add the neurotrophic factor mixture, cell adhesion promoter and antioxidant repair agent to the basal culture medium, mix evenly, and obtain the enhanced basal culture medium after sterilization filtration, and pre-cool for later use;

[0022] S3: Construction of the composite system: The stem cell suspension is added to the enhanced basal culture medium, followed by the addition of the negative hydrogen ion donor, and gently mixed by blowing to obtain a stem cell composition for nerve repair with synergistic effect of negative hydrogen ions.

[0023] Furthermore, in S1, stem cell digestion is performed using a 0.25% trypsin-EDTA digestion solution for 2-4 minutes.

[0024] Furthermore, the centrifugation speed is 800-1200 r / min, and the centrifugation time is 3-5 min.

[0025] Furthermore, in S2, the sterilization filtration is performed using a microporous membrane with a pore size of 0.22 μm.

[0026] Furthermore, the addition of the negative hydrogen ion donor in S3 must be carried out in a sterile and light-protected environment, and the mixed composition must be stored at 4°C for no more than 24 hours.

[0027] This invention addresses key issues in existing nerve repair technologies, such as low stem cell survival rates, insufficient antioxidant capacity, limited nutritional support, and lack of synergistic effects, through precise formulation and synergistic effects of its various components. It utilizes widely available stem cells with low immunogenicity, such as bone marrow mesenchymal stem cells, as the core repair carrier, coupled with a negative hydrogen ion donor. The released negative hydrogen ions form a synergistic antioxidant system with the antioxidant repair agent, doubly eliminating excess reactive oxygen species at the damaged site, alleviating oxidative stress damage to stem cells, and significantly improving stem cell survival efficiency in the in vivo microenvironment. The neurotrophic factor mixture uses a ratio of brain-derived neurotrophic factor, nerve growth factor, and neurotrophic elements, overcoming the limitations of single neurotrophic factors and synergistically regulating nerve cell proliferation, differentiation, and survival, providing comprehensive nutritional support for stem cells to perform their repair functions. A cell adhesion promoter helps stem cells efficiently colonize the nerve damage site, preventing stem cell loss due to insufficient adhesion and ensuring their continued repair function. The basal culture medium provides a stable physicochemical environment for stem cell growth and the synergistic effects of various functional components. Through the synergistic effects of the above-mentioned components in terms of antioxidant protection, nutritional support, adhesion and colonization, and functional induction, this invention effectively solves the technical problems of low stem cell survival rate, insufficient repair efficiency, and poor synergistic effect in the prior art, thereby achieving a more stable and efficient nerve repair effect, fundamentally improving the stem cell repair efficiency, and realizing efficient repair and functional reconstruction of damaged nerve tissue.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] 1. Significantly Enhances Stem Cell Survival and Antioxidant Capacity: This invention constructs a highly efficient antioxidant system by introducing the synergistic effect of negative hydrogen ion donors and novel antioxidant repair agents. This system can effectively eliminate reactive oxygen species at damaged sites, significantly reduce the damage to cells caused by oxidative stress, thereby significantly improving the survival rate and antioxidant stability of stem cells in the in vivo microenvironment, providing a more stable cellular basis for nerve repair.

[0030] 2. Optimizing the cellular microenvironment and enhancing repair effects: The neurotrophic factor mixture and cell adhesion promoter used in this invention work synergistically to further optimize the cellular microenvironment. The neurotrophic factor mixture can synergistically regulate the proliferation, differentiation, and survival of nerve cells, providing comprehensive nutritional support for stem cells to perform their repair functions; the cell adhesion promoter helps stem cells efficiently colonize the nerve injury site, preventing stem cells from being lost due to insufficient adhesion, ensuring their continuous repair function, thereby significantly enhancing the overall effect of nerve repair.

[0031] 3. Improved Stability and Efficiency of Nerve Repair: This invention addresses core issues in existing technologies, such as low stem cell survival rate, insufficient antioxidant capacity, limited nutritional support, and lack of synergistic effects, through the precise proportions and synergistic effects of its various formulation components. This synergistic effect makes the nerve repair process more stable and efficient, fundamentally improving the repair efficiency of stem cells and achieving highly efficient repair and functional reconstruction of damaged nerve tissue, providing a more effective solution for the treatment of nerve injury and neurodegenerative diseases. Attached Figure Description

[0032] Figure 1 The antioxidant repair agent 1 described in this invention 1 HNMR spectrum. Detailed Implementation

[0033] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Preparation Example 1

[0035] Preparation of Antioxidant Repair Agent 1:

[0036] ;

[0037] 5.00 g of compound 1, 2.72 g of compound 2, 6.11 g of potassium carbonate, 0.13 g of cuprous iodide, and 70 mL of DMF were added to a dry reaction vessel. The reaction vessel was purged with nitrogen three times. 0.08 g of pyridine-2-carboxylic acid was added via a syringe. The reaction mixture was heated and stirred at 100 °C for 16 hours. After the reaction was complete, the reaction mixture was cooled to room temperature and then quenched in a saturated ammonium chloride aqueous solution. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined, washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was subjected to silica gel column chromatography, eluted with a mixture of petroleum ether and ethyl acetate, and concentrated under reduced pressure to obtain 4.76 g of compound 3.

[0038] ;

[0039] 4.76 g of compound 3 and 4.44 g of compound 4 were added to a dry reaction vessel, followed by 60 mL of anhydrous THF. The mixture was stirred until the solids were completely dissolved. At room temperature, 4.59 g of N,N-diisopropylethylamine was added to the solution, followed by 7.42 g of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate in portions, ensuring the reaction temperature did not exceed 30 °C. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, the mixture was quenched with a saturated ammonium chloride aqueous solution and extracted with ethyl acetate. The organic layers were combined and washed successively with a saturated sodium bicarbonate aqueous solution and a saturated sodium chloride aqueous solution. The organic layers were dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was subjected to silica gel column chromatography, eluted with a mixture of petroleum ether and ethyl acetate, and concentrated under reduced pressure to obtain 6.98 g of antioxidant repair agent 1.

[0040] Structural assessment:

[0041] Mass spectrometry (MS) of compound 3 [M+H] + =269;

[0042] Mass spectrometry (MS) [M+H] of antioxidant repair agent 1 + =501;

[0043] Antioxidant Repair Agent 1 1 HNMR such as Figure 1 As shown.

[0044] Preparation Examples 2-4

[0045] In Preparation Examples 2-4, antioxidant repair agents 2-4 were prepared sequentially, following the same preparation method as in Preparation Example 1, except that compound 2 was replaced, while the rest remained the same. The specific structures and mass spectrometry (MS[M+H]) spectra of antioxidant repair agents 2-4 are shown below. + The structure of compound 2 is shown in Table 1.

[0046] Table 1

[0047]

[0048] Example 1

[0049] Preparation of a stem cell composition for nerve repair with synergistic effects of negative hydrogen ions:

[0050] 1. Raw material components:

[0051] Stem cells: 8 units (for OriCell) ® Adult bone marrow mesenchymal stem cells (purchased from: Cyagen (Suzhou) Biotechnology Co., Ltd.)

[0052] Negative hydrogen ion donor: 0.08 parts (reduced nicotinamide adenine dinucleotide);

[0053] Neurotrophic factor mixture: 0.5 parts (composed of brain-derived neurotrophic factor, nerve growth factor and neurotrophin-3 in a mass ratio of 2:1:1, purchased from: Yisheng Biotechnology (Shanghai) Co., Ltd.);

[0054] Cell adhesion promoter: 0.03 parts (laminin, purchased from: Yisheng Biotechnology (Shanghai) Co., Ltd.);

[0055] Antioxidant repair agent: 0.1 parts (antioxidant repair agent 1 prepared in preparation example 1);

[0056] Basic culture medium: 85 portions (DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin antibiotics).

[0057] 2. Preparation method:

[0058] S1. Stem Cell Acquisition and Culture: Bone marrow mesenchymal stem cells were harvested and cultured in vitro at 37°C in a 5% CO2 incubator using the aforementioned basal culture medium. When the cells reached the 4th passage, they were digested with 0.25% trypsin-EDTA digestion solution for 3 minutes. After digestion, basal culture medium was added to terminate the digestion. The cell suspension was collected and washed three times with PBS buffer. The washed cells were then centrifuged at 1000 rpm for 4 minutes. The supernatant was discarded, and the pellet was resuspended in basal culture medium, adjusting the stem cell concentration to 3 × 10⁻⁶ cells / min. 6 The cells / mL were used to prepare a homogeneous stem cell suspension;

[0059] S2. Preparation of substrate solution: The mixture of neurotrophic factors, cell adhesion promoters, and antioxidant repair agents were added to the basal culture medium and stirred at room temperature for 30 min to ensure that all components were fully dissolved and mixed evenly. Subsequently, the mixture was sterilized by filtration through a microporous membrane with a pore size of 0.22 μm. After filtration, the resulting enhanced basal culture medium was pre-cooled at 4°C for later use.

[0060] S3. Construction of the composite system: In a sterile operating table, the stem cell suspension prepared in step S1 was slowly added to the enhanced basal culture medium pre-cooled in step S2, while gently agitating the mixture to ensure uniform dispersion of the stem cells. Subsequently, under light-protected conditions, the correct amount of negative hydrogen ion donor was added, and gentle agitation was continued for 10 minutes to ensure thorough mixing of all components, thus obtaining a stem cell composition for neural repair with synergistic effects of negative hydrogen ions.

[0061] Examples 2-4

[0062] The preparation of a stem cell composition for neural repair with synergistic effect of negative hydrogen ions is carried out by referring to the preparation method of Example 1, except that the antioxidant repair agent is replaced in sequence with antioxidant repair agent 2-antioxidant 4 prepared in Preparation Examples 2-4, and other operations are the same as in Example 1.

[0063] Comparative Example 1

[0064] The preparation of a stem cell composition for nerve repair with synergistic effect of negative hydrogen ions is carried out according to the preparation method of Example 1, except that the antioxidant repair agent is replaced with vitamin C, and other operations are the same as in Example 1.

[0065] Comparative Example 2

[0066] The preparation of a stem cell composition for nerve repair with synergistic effect of negative hydrogen ions is carried out according to the preparation method of Example 1, except that the antioxidant repair agent is replaced with vitamin E, and other operations are the same as in Example 1.

[0067] Comparative Example 3

[0068] The preparation of a stem cell composition for neural repair with synergistic effect of negative hydrogen ions is carried out according to the preparation method of Example 1, without the addition of antioxidant repair agent, and other operations are the same as in Example 1.

[0069] Comparative Example 4

[0070] The preparation of a stem cell composition for nerve repair with synergistic effect of negative hydrogen ions is carried out according to the preparation method of Example 1, except that the neurotrophic factor mixture is not added, and other operations are the same as in Example 1.

[0071] Performance testing:

[0072] 1. In vivo neural repair efficacy test (rat middle cerebral artery embolism MCAO model)

[0073] 1.1. Test Objective:

[0074] To verify the composition's repair effect and functional reconstruction effect on damaged nerve tissue in vivo.

[0075] 1.2. Materials and Instruments:

[0076] Test samples: Compositions prepared in Examples 1-4 and Comparative Examples 1-4 (stored at 4°C, used within 24 hours);

[0077] Experimental animals: SPF-grade SD rats (weighing 220-250g), 10 rats per group;

[0078] Reagents: Chloral hydrate, physiological saline, 4% paraformaldehyde, β-tubulin III antibody (neural regeneration marker), GFAP antibody (glial scar marker), DAPI staining solution;

[0079] Instruments: stereotaxic apparatus, surgical instruments, cryostat, fluorescence microscope, behavioral scoring device.

[0080] 1.3. Test Procedure

[0081] (1) Animal model preparation:

[0082] Rats were anesthetized intraperitoneally with 10% chloral hydrate (3.5 mL / kg), fixed in a supine position, and the right common carotid artery, external carotid artery, and internal carotid artery were separated. A nylon suture was inserted to embolize the right middle cerebral artery, and the suture was removed after 2 hours of blood flow obstruction to establish a transient cerebral ischemia-reperfusion injury model (simulating neurodegenerative injury). Postoperatively, the rats were observed for neurological deficit symptoms (such as hemiplegia and balance disorders), and rats that successfully modeled the injury were selected for subsequent experiments.

[0083] (2) Administration method:

[0084] 24 hours after successful modeling, 5 μL of test sample (stem cell concentration 3 × 10⁻⁶) was injected into the ischemic striatal region of the rat using a stereotaxic instrument. 6 (cells / mL), while the control group was injected with an equal volume of physiological saline or pure stem cell suspension.

[0085] (3) Behavioral assessment (neurological function recovery):

[0086] Motor function of rats was assessed using the Longa neurological function scoring system at 7, 14, and 28 days after drug administration.

[0087] 0 points: No neurological deficit;

[0088] 1 point: The left forelimb cannot be fully extended;

[0089] 2 points: Turning in a circle to the left while walking;

[0090] 3 points: Leaning to the left while walking;

[0091] 4 points: Unable to walk independently, impaired consciousness.

[0092] (4) Histological examination:

[0093] 28 days after drug administration, rats were anesthetized and their hearts were perfused with 4% paraformaldehyde. Brain tissue was then collected, fixed, and prepared into frozen sections (10 μm thick).

[0094] Immunofluorescence staining: Add β-tubulin III antibody (primary antibody dilution ratio 1:200) and GFAP antibody (primary antibody dilution ratio 1:200), incubate overnight at 4°C, incubate with fluorescent secondary antibody for 1 hour, and stain the nucleus with DAPI;

[0095] Fluorescence microscopy observation: The number of β-tubulin III positive cells (indicator of nerve regeneration) and the area of ​​GFAP positive regions (indicator of glial scar formation) were counted. The results are shown in the table below.

[0096] Table 2

[0097]

[0098] Table 2 shows that the neurological function scores of rats in Examples 1 to 4 gradually decreased over time, indicating gradual recovery of neurological function. The scores were also low at 28 days after administration, suggesting good nerve repair. Simultaneously, the number of β-tubulin III positive cells in these groups showed an increasing trend, indicating good nerve regeneration; while the area of ​​GFAP positive regions showed a decreasing trend, indicating that glial scar formation was inhibited. In contrast, the neurological function scores of Comparative Examples 1 to 4 and the control group decreased more slowly, with less obvious neurological function recovery, fewer β-tubulin III positive cells, poorer nerve regeneration, and larger GFAP positive regions, indicating more glial scar formation. This indicates that the compositions in Examples 1 to 4 demonstrate significant superiority in in vivo nerve repair, effectively promoting nerve regeneration and inhibiting glial scar formation, thereby better improving functional recovery after nerve injury.

[0099] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A stem cell composition for neural repair with synergistic effects of negative hydrogen ions, characterized in that, By weight, it consists of the following components: 5-10 parts stem cells, 0.05-0.1 parts negative hydrogen ion donor, 0.1-1 parts neurotrophic factor mixture, 0.02-0.05 parts cell adhesion promoter, 0.05-0.2 parts antioxidant repair agent, and 80-90 parts basal culture medium. The antioxidant repair agent is a compound shown in Formula 1: ； In Formula 1, R1 is at least one of methyl, ethyl, methoxy, and nitro.

2. The stem cell composition for neural repair with synergistic enhancement of negative hydrogen ions according to claim 1, characterized in that, The stem cells mentioned are bone marrow mesenchymal stem cells.

3. The stem cell composition for neural repair with synergistic enhancement of negative hydrogen ions according to claim 1, characterized in that, The negative hydrogen ion donor is at least one of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate.

4. The stem cell composition for neural repair with synergistic enhancement of negative hydrogen ions according to claim 1, characterized in that, The neurotrophic factor mixture consists of brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3, with a mass ratio of 2:1:

1.

5. The stem cell composition for neural repair with synergistic enhancement of negative hydrogen ions according to claim 1, characterized in that, The basal culture medium is DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin antibiotics.

6. The stem cell composition for neural repair with synergistic enhancement of negative hydrogen ions according to claim 1, characterized in that, The antioxidant repair agent is any one of the following compounds: 。 7. The stem cell composition for neural repair with synergistic enhancement of negative hydrogen ions according to claim 1, characterized in that, The cell adhesion promoter is laminin.

8. A method for preparing a stem cell composition for neural repair with synergistic effects of negative hydrogen ions as described in any one of claims 1-7, characterized in that, Includes the following steps: S1. Stem cell acquisition and culture: The stem cells were collected and cultured in the basal culture medium at 37°C and 5% CO2 in an incubator to the 3rd-5th generation. After digestion and collection, the stem cells were washed 2-3 times with PBS buffer, centrifuged, washed again, and resuspended in the basal culture medium to adjust the stem cell concentration to 1×10⁻⁶. 6 -5×10 6 cells / mL, to prepare a stem cell suspension; S2. Preparation of substrate solution: The neurotrophic factor mixture, cell adhesion promoter and antioxidant repair agent are added to the basal culture medium, mixed evenly, and filtered after sterilization to obtain the enhanced basal culture medium, which is then pre-cooled for later use. S3. Construction of the composite system: The stem cell suspension is added to the enhanced basal culture medium, followed by the addition of the negative hydrogen ion donor, and gently mixed by blowing to obtain a stem cell composition for nerve repair with synergistic effect of negative hydrogen ions.

9. The method for preparing a stem cell composition for neural repair with synergistic enhancement of negative hydrogen ions according to claim 8, characterized in that, In S1, stem cell digestion is performed using 0.25% trypsin-EDTA digestion solution for 2-4 minutes. The centrifugation speed is 800-1200 r / min, and the centrifugation time is 3-5 min.

10. The method for preparing a stem cell composition for neural repair with synergistic enhancement of negative hydrogen ions according to claim 8, characterized in that, The sterilization filtration in S2 uses a microporous membrane with a pore size of 0.22 μm for filtration; The addition of the negative hydrogen ion donor in S3 must be carried out in a sterile and light-protected environment, and the mixed composition must be stored at 4°C for no more than 24 hours.

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