Mesenchymal stem cell based on intermittent gradient hypoxia pretreatment, preparation method of mesenchymal stem cell and application of mesenchymal stem cell in medicine for treating premature ovarian failure

Abstract

The invention discloses a mesenchymal stem cell based on intermittent gradient hypoxia pretreatment, a preparation method of the mesenchymal stem cell and application of the mesenchymal stem cell in a medicine for treating premature ovarian failure, and relates to the technical field of biological medicine. The preparation method of the mesenchymal stem cells based on intermittent gradient low-oxygen pretreatment comprises the following steps: S1, separating primary mesenchymal stem cells and inoculating the primary mesenchymal stem cells in a serum-free alpha-MEM complete medium; s2, the primary mesenchymal stem cells are subjected to intermittent gradient low oxygen-normal oxygen alternate pretreatment culture in a serum-free alpha-MEM complete medium; s3, carrying out cell passage and amplification to harvest subcultured mesenchymal stem cells; the dynamic hypoxia pretreatment can significantly improve the ability of MSC to adapt to the ovarian special pathological environment, maintain the young state and functionality of MSC cells, introduce antioxidant components, significantly reduce MSC oxidative damage, combine a multi-way administration mode, optimize MSC homing and repairing efficiency, and significantly improve the comprehensive effect of ovarian function and fertility recovery.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a mesenchymal stem cell based on intermittent gradient hypoxia pretreatment, its preparation method, and its application in drugs for treating premature ovarian failure. Background Technology

[0002] In existing technologies, mesenchymal stem cells (MSCs) have been widely used in tissue engineering and regenerative medicine due to their tissue repair and regeneration potential. Several patents have disclosed the use of MSCs cultured under constant hypoxia (e.g., 5% or lower oxygen concentration) conditions for the treatment of musculoskeletal diseases such as osteoarthritis. Compared to normoxic conditions (NR-MSCs), MSCs cultured under constant hypoxia exhibit significant advantages in delaying cell senescence, enhancing cell proliferation and differentiation, improving migration ability, and increasing the secretion of pro-angiogenic factors (such as VEGF and HGF), thereby promoting bone and cartilage repair. However, these MSCs cultured under constant hypoxia are mainly used in the field of musculoskeletal diseases such as osteoarthritis and bone defects, and their application in reproductive system-related diseases such as premature ovarian failure (POF) remains relatively limited.

[0003] Specifically, existing MSC products and preparation methods on the market have the following drawbacks:

[0004] 1. The preparation method uses a single culture condition, which is difficult to adapt to the pathological environment of the ovary:

[0005] Premature ovarian failure (POF) induced by chemotherapy drugs such as cyclophosphamide involves a complex pathological microenvironment of alternating hypoxia and reoxygenation within the ovary. While constant hypoxic culture conditions can initially activate the HIF-1α pathway in mesenchymal stem cells (MSCs), prolonged exposure to a single hypoxic environment can easily lead to decreased tolerance of MSCs to hypoxia, failing to fully mimic the pathological characteristics (hypoxia-reoxygenation alternation) of actual ovarian tissue. Therefore, current techniques are insufficient to effectively activate the antioxidant stress and regenerative repair capabilities necessary for MSCs under POF pathological conditions.

[0006] 2. Limited indications, lack of specific design for POF:

[0007] Existing hypoxic MSC culture techniques are mostly designed for the repair needs of osteoarthritis or other osteochondral-related diseases, lacking optimized pretreatment protocols for the specific pathological characteristics of ovarian tissue, such as local inflammation, ischemia, reperfusion injury, and endocrine disorders. Therefore, they are difficult to directly apply to the clinical scenario of premature ovarian failure.

[0008] In summary, existing MSC products and preparation methods have significant shortcomings in the field of drugs for the treatment of premature ovarian failure (POF), particularly lacking intermittent, dynamic hypoxia-noroxic pretreatment culture adapted to the unique pathological environment of POF. Therefore, there is an urgent need to develop a mesenchymal stem cell (MSC) preparation method based on intermittent gradient hypoxia pretreatment and its application in drugs for treating POF, in order to effectively improve the clinical efficacy of MSCs in the preparation of drugs for treating POF. Summary of the Invention

[0009] In view of this, the present invention addresses the deficiencies of existing technologies. Its main objective is to provide a method for preparing mesenchymal stem cells (MSCs) based on intermittent gradient hypoxia pretreatment and their application in drugs for treating premature ovarian failure. The dynamic hypoxia pretreatment significantly improves the ability of MSCs to adapt to the special pathological environment of the ovary, shortens the acclimatization cycle, maintains the youthfulness and functionality of MSCs, introduces antioxidant components to significantly reduce oxidative damage to MSCs, combines multiple drug delivery routes to optimize MSC homing and repair efficiency, evaluates the efficacy of MSCs through multi-dimensional and multi-level indicators, objectively and comprehensively verifies the efficacy of MSCs, and establishes a functional quality control indicator system to ensure batch stability of MSCs, thereby significantly improving the overall effect of ovarian function and fertility recovery.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A method for preparing mesenchymal stem cells based on intermittent gradient hypoxia pretreatment includes the following steps:

[0012] S1. Isolation of primary mesenchymal stem cells and inoculation in serum-free α-MEM complete medium;

[0013] S2. Primary mesenchymal stem cells were cultured in serum-free α-MEM complete medium under intermittent gradient hypoxia-noroxic alternating pretreatment.

[0014] S3. Cell passage and expansion: Harvest passaged mesenchymal stem cells.

[0015] As a preferred embodiment, the intermittent gradient hypoxia-noroxic alternating pretreatment culture in step S2 specifically involves:

[0016] On the first day, the cells were cultured for 24 hours under normal aerobic conditions: 21% O2, 5% CO2, and 37°C to activate initial proliferation.

[0017] Starting the next day, an intermittent alternation method was used for oxygen tension switching culture, namely: hypoxia condition: oxygen concentration of 2%–10% O2; normoxic condition: oxygen concentration of 21% O2; the hypoxia condition and normoxic condition were alternated every 12–36 hours, and the cycle was repeated 6–12 times.

[0018] As a preferred embodiment, the serum-free α-MEM complete culture medium in step S1 comprises: α-MEM basal culture medium: 90%–95% by volume; human platelet lysate (PLTGold): 2%–7% by volume; nutrient additive (Nutri-Add): 0.5%–3% by volume; and antioxidant.

[0019] As a preferred embodiment: the antioxidant in step S1 is selected from N-acetylcysteine ​​(NAC) with a final concentration of 1-5 mM; glutathione (GSH) with a final concentration of 0.1-1 mM; vitamin C with a final concentration of 100-200 μM; lipoic acid with a final concentration of 50-100 μM and coenzyme Q10 with a final concentration of 2-20 μM.

[0020] As a preferred embodiment, the antioxidants in step S1 include N-acetylcysteine ​​(NAC) at a final concentration of 1-5 mM and coenzyme Q10 (CoQ10) at a final concentration of 2-20 μM.

[0021] As a preferred embodiment, the cell passage and expansion in step S3 specifically includes the following steps:

[0022] S31. When the cell confluence reaches 80%, digest with 0.25%-0.5% trypsin / EDTA, and then neutralize with 3-5 times the volume of serum-free α-MEM complete medium.

[0023] Centrifuge at 300g-500g for 5-10 minutes, discard the supernatant, and resuspend the cells;

[0024] S33. Passage the cells at a ratio of 1:3–1:4 and continue intermittent alternating hypoxia-noroxic culture, i.e.: hypoxia condition: oxygen concentration of 2%–10% O2; normoxic condition: oxygen concentration of 21% O2; alternate between hypoxia and normoxic conditions every 12–36 hours, for 6–12 cycles; harvest P4 or P5 generation mesenchymal stem cells.

[0025] As a preferred embodiment, step S1 specifically includes the following steps:

[0026] S11. Selection of primary mesenchymal stem cell sources and isolation of primary mesenchymal stem cells;

[0027] S12. Cell Seeding: Primary mesenchymal stem cells were resuspended in serum-free α-MEM complete medium and seeded at 5,000 cells / cm³. 2 Density seeding was carried out in the culture platform.

[0028] A method for preparing mesenchymal stem cells using intermittent gradient hypoxia pretreatment.

[0029] The application of mesenchymal stem cells in a drug for treating premature ovarian failure, wherein the mesenchymal stem cells are prepared as a drug for treating premature ovarian failure.

[0030] As a preferred option, the administration method of the drug is selected from any one of subcapsular injection of the ovary, tail vein injection, and ovarian artery injection.

[0031] Compared with the prior art, the present invention has significant advantages and beneficial effects. Specifically, as can be seen from the above technical solution,

[0032] 1. Dynamic hypoxia pretreatment significantly improves the ability of MSCs to adapt to the special pathological environment of the ovary:

[0033] The dynamic, intermittent gradient hypoxia-noroxic alternating culture method with 2%–10% O2 concentration effectively simulated the pathological characteristics of "hypoxia-reoxygenation" in ovarian tissue after chemotherapy, activated the HIF-1α signaling pathway and related antioxidant and anti-apoptotic mechanisms, and significantly improved the survival rate and functional activity of MSCs in the ovary.

[0034] 2. Shorten the domestication cycle to maintain the youthfulness and functionality of MSC cells:

[0035] Limiting the MSC domestication process to the P2–P5 generation avoids cell senescence and telomere shortening caused by long-term expansion, significantly improves the paracrine capacity and repair efficiency of MSCs, and ensures the high efficiency and stable biological activity of MSCs.

[0036] 3. Introducing antioxidants significantly reduces oxidative damage to MSCs:

[0037] The addition of N-acetylcysteine ​​(NAC) and coenzyme Q10 (CoQ10) as antioxidants during the culture process effectively alleviates oxidative stress generated during the alternating hypoxia-reoxygenation culture process, significantly reduces cell damage, and maintains the stability and high efficiency of cell therapy preparations.

[0038] 4. Combining multiple drug delivery routes to optimize MSC homing and repair efficiency:

[0039] This study innovatively compared three different MSC infusion routes: subcapsular injection, tail vein injection, and ovarian artery injection. The results showed that, through comprehensive evaluation of multiple indicators, including serum hormone levels, follicle count in HE-sectioned ovarian tissue, observation of fertility outcomes, and expression of molecular markers (such as pro-angiogenic factors and anti-inflammatory factors), the combined administration strategy significantly improved the actual therapeutic effect of MSCs and the ovarian repair capacity.

[0040] 5. The evaluation indicators are multi-dimensional and multi-level, objectively and comprehensively verifying the efficacy of MSC:

[0041] In the animal model evaluation, a mouse model of premature ovarian failure was induced by CTX for 15 consecutive days (highly simulating the clinical chemotherapy scenario); on day 21, a single infusion of 2×10^6 PD-MSCs was performed, and a positive control (estradiol valerate) was established to ensure the effectiveness of the model; on day 49, a comprehensive evaluation was carried out on serum hormones (FSH, E2, AMH, etc.), follicle count in HE slides, fertility assessment (pregnancy rate, number of fetuses), and various molecular indicators (VEGF, HGF, TNF-α, IL-6, etc.) to achieve a multidimensional and comprehensive evaluation of cell and animal models, effectively overcoming the misjudgment of efficacy caused by single or one-sided evaluation indicators in the past, and enhancing the objectivity and credibility of the efficacy.

[0042] 6. A functional quality control indicator system to ensure MSC batch stability:

[0043] A multifunctional indicator detection system was introduced, including cell phenotype (CD73, CD90, CD105 expression), oxygen stress pathway molecules (HIF-1α, CXCR4), and cell metabolism indicators (OCR / ECAR ratio), which ensured a high degree of consistency in the quality and therapeutic function of the prepared MSCs between batches, effectively solving the problems of the traditional MSC quality evaluation system being weak and unstable.

[0044] 7. Significantly improves overall ovarian function and fertility recovery:

[0045] Animal models have validated that the MSC treatment group of this patent is significantly superior to single-pathway drug administration or conventional hypoxic / noroxic culture MSC treatment protocols in multiple indicators, including endocrine hormone regulation, follicle structure recovery, improved fertility, reduced inflammatory factors, and ovarian angiogenesis. This indicates that the innovative protocol not only achieves a technological breakthrough in cell preparation but also has significant advantages in therapeutic effects, demonstrating the technological advancement and clinical application prospects of the dual dimensions of cell and animal evaluation.

[0046] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0047] Figure 1 Cell observation of MSC cells under different treatment methods according to the present invention;

[0048] Figure 2 The viability of MSCs after cryopreservation and thawing under different processing methods of this invention;

[0049] Figure 3 Evaluation of MSC chemotactic migration ability under different treatment methods of the present invention;

[0050] Figure 4This is a comparison chart of the chemotactic migration ability of MSCs under different treatment methods of the present invention;

[0051] Figure 5 This is a schematic diagram showing the changes in MSC oxygen tolerance under different treatment methods according to the present invention;

[0052] Figure 6 This is a schematic diagram showing the changes in MSC dryness index under different treatment methods of the present invention;

[0053] Figure 7 This is a schematic diagram illustrating the changes in MSC immune function under different treatment methods according to the present invention;

[0054] Figure 8 This is a schematic diagram showing the changes in MSC proliferation capacity under different treatment methods according to the present invention;

[0055] Figure 9 This is a schematic diagram showing the amount of VEGF secreted in the MSC culture supernatant under different treatment methods of the present invention;

[0056] Figure 10 This is a schematic diagram illustrating the cell viability test of MSCs under different treatment methods induced by hydrogen peroxide during hydrogen peroxide oxidation.

[0057] Figure 11 This is a schematic diagram of three drug administration routes for the mouse POF model of the present invention;

[0058] Figure 12 This is a schematic diagram illustrating the changes in FSH concentration in a mouse POF model after treatment under different cell types and administration routes according to the present invention.

[0059] Figure 13 This is a schematic diagram illustrating the reproductive rate of a mouse POF model after treatment with different cell types and administration routes according to the present invention.

[0060] Figure 14 These are images showing the morphological characteristics of mice giving birth to pups under different cell types and drug administration routes according to the present invention. Detailed Implementation

[0061] The present invention is as follows Figures 1 to 14 As shown, a method for preparing mesenchymal stem cells based on intermittent gradient hypoxia pretreatment includes the following steps:

[0062] S1. Isolation of primary mesenchymal stem cells and inoculation in serum-free α-MEM complete medium;

[0063] S2. Primary mesenchymal stem cells were cultured in serum-free α-MEM complete medium under intermittent gradient hypoxia-noroxic alternating pretreatment.

[0064] In step S2, the culture medium should be replaced with fresh serum-free α-MEM complete medium every 48 hours during the culture process;

[0065] S3. Cell passage and expansion: Harvest passaged mesenchymal stem cells.

[0066] The specific steps of intermittent gradient hypoxia-noroxic alternating pretreatment culture in step S2 are as follows:

[0067] On the first day, the cells were cultured for 24 hours under normal aerobic conditions: 21% O2, 5% CO2, and 37°C to activate initial proliferation.

[0068] Starting the next day, an intermittent alternation method was used for oxygen tension switching culture, namely: hypoxia condition: oxygen concentration of 2%–10% O2; normoxic condition: oxygen concentration of 21% O2; the hypoxia condition and normoxic condition were alternated every 12–36 hours, and the cycle was repeated 6–12 times.

[0069] The serum-free α-MEM complete culture medium in step S1 includes: α-MEM basal medium: 90%–95% by volume; human platelet lysate (PLTGold): 2%–7% by volume; nutrient additive (Nutri-Add): 0.5%–3% by volume; and antioxidants.

[0070] In step S1, the antioxidants are selected from N-acetylcysteine ​​(NAC) at a final concentration of 1-5 mM; glutathione (GSH) at a final concentration of 0.1-1 mM; vitamin C at a final concentration of 100-200 μM; lipoic acid at a final concentration of 50-100 μM; and coenzyme Q10 at a final concentration of 2-20 μM. Antioxidants are added to reduce ROS generation during low-oxygen culture.

[0071] The antioxidants in step S1 include N-acetylcysteine ​​(NAC) at a final concentration of 1-5 mM and coenzyme Q10 (CoQ10) at a final concentration of 2-20 μM.

[0072] Basic culture medium: α-MEM, 90%–95% (v / v) provides the essential nutrients required for cell growth;

[0073] Human platelet lysate PLTGold, 2-7% (v / v): a substitute for fetal bovine serum, providing growth factors and cytokines required for cell growth, improving the safety and clinical translation of MSCs;

[0074] Nutri-Add, 0.5%–3% (v / v): Provides vitamins, amino acids and trace elements to optimize MSC metabolism;

[0075] Antioxidants: N-acetylcysteine ​​(NAC), final concentration 1–5 mM; significantly scavenges reactive oxygen species (ROS) generated during culture, improving cell viability and stability; Coenzyme Q10 (CoQ10), final concentration 5–20 μM; improves mitochondrial function, reduces cell damage caused by oxidative stress, and improves cell quality.

[0076] The cell passage and expansion in step S3 specifically includes the following steps:

[0077] S31. When the cell confluence reaches 80%, digest with 0.25%-0.5% trypsin / EDTA, and then neutralize with 3-5 times the volume of serum-free α-MEM complete medium.

[0078] Centrifuge at 300g-500g for 5-10 minutes, discard the supernatant, and resuspend the cells;

[0079] S33. Passage the cells at a ratio of 1:3–1:4 and continue intermittent alternating hypoxia-noroxic culture, i.e.: hypoxia condition: oxygen concentration of 2%–10% O2; normoxic condition: oxygen concentration of 21% O2; alternate between hypoxia and normoxic conditions every 12–36 hours, for 6–12 cycles; harvest P4 or P5 generation mesenchymal stem cells.

[0080] Simulating the hypoxic-reoxygenated environment within the ovary, HIF-1α and CXCR4 are activated, enhancing the antioxidant, anti-apoptotic, and migration capabilities of MSCs.

[0081] Step S1 specifically includes the following steps:

[0082] S11. Selection of primary mesenchymal stem cell sources and isolation of primary mesenchymal stem cells;

[0083] S12. Cell Seeding: Primary mesenchymal stem cells were resuspended in serum-free α-MEM complete medium and seeded at 5,000 cells / cm³. 2 Density seeding was carried out in the culture platform.

[0084] A method for preparing mesenchymal stem cells using intermittent gradient hypoxia pretreatment.

[0085] The application of a mesenchymal stem cell in a drug for treating premature ovarian failure, wherein the mesenchymal stem cells are prepared as a drug for treating premature ovarian failure.

[0086] The drug can be administered via any one of the following methods: subcapsular injection, tail vein injection, or ovarian artery injection.

[0087] Current technologies offer only one route of administration, limiting the homing and retention of MSCs in the ovary.

[0088] Existing MSC treatment methods typically employ single-dose administration methods such as tail vein injection or local single-point injection, which fail to fully utilize the strong homing potential and local retention advantages of MSC cells. This results in a limited number of effective cells entering the ovarian tissue, thereby limiting the therapeutic effect.

[0089] The primary mesenchymal stem cells used in this application are derived from any one of the following: umbilical cord Wharton's Jelly, placental MSCs (P-MSC), amniotic MSCs (A-MSC), adipose MSCs (AD-MSC), or bone marrow MSCs (BM-MSC).

[0090] The culture platform can employ 2D cell factories, three-dimensional suspension culture of microcarriers, or bioreactor systems; controllable stirred microcarrier systems (such as Cytodex-3) can be used to conduct alternating oxygen tension culture in a hypoxic chamber to expand yield.

[0091] Bioreactor system: A closed bioreactor equipped with an oxygen tension control module is used for intermittent low-oxygen stimulation culture to achieve full automation of the process.

[0092] Treatment methods and administration routes: Local gel sustained-release carrier infusion can also be used, in which hypoxia-pretreated MSCs are mixed into the gelatin-sodium alginate gel system and slowly released to the ovarian surface or local area to achieve local continuous exposure.

[0093] Compared with the prior art, this application has the following advantages:

[0094] 1. Dynamic hypoxia activation, simulating real stress environment in vivo.

[0095] Traditional hypoxic MSCs are often cultured at a constant hypoxic concentration, failing to simulate the intermittent hypoxia and reoxygenation process experienced by ovarian tissue after chemotherapy damage. This invention employs a culture strategy that alternates between "2%–5% hypoxia and normoxic (21%)", more accurately simulating the ischemia / reperfusion pathological environment. This allows for the early activation of the MSCs' response mechanism to changes in oxygen tension in vitro, thereby improving their in vivo survival and functional stability.

[0096] 2. Promotes both angiogenesis and homing ability.

[0097] The intermittent hypoxic MSCs prepared by this invention have significantly upregulated expression on key signal axes such as chemotactic properties and functional properties. Compared with normoxic conditions, they have stronger pro-angiogenic and chemotactic migration capabilities and can quickly home to damaged ovarian sites, achieving more efficient repair coverage.

[0098] 3. Lower oxidative stress injury and metabolic adaptation burden

[0099] Compared to the risk of apoptosis and ROS accumulation caused by constant extremely low oxygen (1–2%), the present invention uses "short-cycle alternating hypoxia" to activate cell repair pathways while avoiding metabolic imbalance, cell stress or proliferation inhibition caused by long-term hypoxia, effectively prolonging the functional cycle of MSCs.

[0100] 4. Balancing cell rejuvenation with a low-passage strategy

[0101] This invention selects P1 generation for pretreatment and completes the preparation by P4–P5. Compared with traditional P5–P7 cells, they have lower senescence markers, higher differentiation potential and stable phenotype, providing higher quality cell preparations for clinical applications, while reducing the risk of genetic drift caused by long-term culture.

[0102] 5. Design of a dual-factor antioxidant-enhanced culture medium

[0103] The formulation incorporates N-acetylcysteine ​​(NAC) and coenzyme Q10 for synergistic antioxidant effects, along with nutrient factors such as PLTGold, enabling MSCs to maintain mitochondrial function and gene stability during hypoxic stress, which is significantly superior to traditional culture systems without added antioxidants.

[0104] 6. Multiple routes of combined drug administration overcome the limitations of single routes.

[0105] Unlike existing methods that rely solely on tail vein or ovarian injection, this invention compares and evaluates three pathways—tail vein, ovarian artery, and subcapsular ovarian injection—in animal models. It found that differential drug administration can improve cell homing efficiency and local repair effects, especially showing significant advantages in multiple dimensions such as follicle number, estrogen levels, and reproductive outcomes.

[0106] 7. Match a multimodal evaluation system to verify the repair effect.

[0107] By constructing a CTX-induced POF model and combining multiple dimensions of evaluation, including serum hormone (E2 / FSH), follicle count, reproductive rate, and histological HE sections, the results showed that the hypoxia-pretreated MSC group was superior to the positive drug control (estradiol valerate) and the untreated MSC group in multiple indicators, fully demonstrating its therapeutic advantages.

[0108] 8. Adaptable to clinical processes, facilitating GMP-compliant large-scale production.

[0109] This method relies on a two-dimensional cell factory platform, avoiding three-dimensional carriers or highly complex bioreactors. The process flow is clear, the steps are standardized, the consumables are universal, and it facilitates large-scale transformation and IND application.

[0110] The hypoxia-pretreated MSCs described in this invention can be further used to extract exosomes (EVs) as cellular component substitutes, and can achieve the basic effect of repairing POF through tail vein infusion.

[0111] Example 1: A method for preparing mesenchymal stem cells based on intermittent gradient hypoxia pretreatment, comprising the following steps:

[0112] S1. Isolation of primary mesenchymal stem cells and inoculation in serum-free α-MEM complete medium:

[0113] Umbilical cord tissue collection and processing

[0114] 1. Select commercially available umbilical cord tissue (20–30 cm).

[0115] 2. Immediately place in serum-free DMEM transport solution containing 100 U / mL penicillin and 100 μg / mL streptomycin, store at 4°C, and transfer to the laboratory for processing within 6 hours.

[0116] 3. Remove the umbilical cord skin and blood vessels, and use sterile scissors to cut the Wharton's jelly into pieces approximately 1mm in size. 3 Organizational block.

[0117] Primary culture and isolation of mesenchymal stem cells

[0118] 1. Distribute the tissue blocks evenly in a 6cm culture dish and culture them using the adherent culture method. Add 2–3 mL of complete culture medium (see composition below) to cover the tissue blocks.

[0119] 2. Initially, the "adherent + undisturbed" strategy was adopted, and the plants were placed in an incubator at 37℃ and 5% CO2 for 72 hours for static incubation.

[0120] 3. Observe the cells crawling out and showing a typical spindle shape on days 7–10. Start passage when 70–80% of cells have reached confluence.

[0121] Complete culture medium composition and preparation

[0122] This invention uses serum-free MSC complete culture medium, the formulation of which is as follows (total volume calculated as 100 mL):

[0123] α-MEM basal medium: 90–95 mL;

[0124] Human platelet lysate (PLTGold): 2-7 mL;

[0125] Nutri-Add: 0.5–3 mL;

[0126] N-acetylcysteine ​​(NAC): Final concentration 1-5 mM;

[0127] Coenzyme Q10 (CoQ10): Final concentration 2-20 μM;

[0128] All liquid components were filtered through a 0.22μm sterile filter membrane before use and were prepared immediately.

[0129] S2. Primary mesenchymal stem cells were cultured in serum-free α-MEM complete medium under intermittent gradient hypoxia-noroxic alternating pretreatment:

[0130] 1. Select P1 generation cells and culture them in a cell factory at a rate of 5–8 × 10⁶ cells / year. 4 pcs / cm 2 Planting density.

[0131] 2. On the first day, the cells were cultured under normal aerobic conditions (21% O2, 5% CO2, 37℃) for 24 hours to activate the initial proliferation.

[0132] 3. Starting the next day, oxygen tension switching culture will be carried out using an intermittent alternation method:

[0133] Low oxygen conditions: 2–5% O2, for 48 hours;

[0134] Normal oxygen conditions: 21% O2, for 24 hours;

[0135] This is considered one cycle, and 2–4 cycles are switched continuously (i.e., the total culture time is 6–12 days).

[0136] 4. Replace the culture medium with fresh complete medium every 48 hours during the culture process.

[0137] S3. Cell passage and expansion: Harvest passaged mesenchymal stem cells.

[0138] Cell passage and expansion

[0139] 1. When cell confluence reaches 80%, digest with 0.25% trypsin / EDTA, and then neutralize with 3 times the volume of culture medium after digestion.

[0140] Centrifuge at 2.300g for 5 minutes, discard the supernatant, and resuspend the cells.

[0141] 3. Passage at a ratio of 1:3–1:4 and continue intermittent alternating hypoxia-noroxic culture.

[0142] 4. Harvest P4 or P5 generation cells for subsequent animal model treatment in early ovarian screening.

[0143] Quality control and functional testing

[0144] 1. Phenotypic identification: Flow cytometry was used to detect the positive expression rate of CD73, CD90, and CD105 (≥95%), and the negative expression rate of CD45 / CD34 (<2%).

[0145] 2. Functional indicators, such as oxygen tolerance, anti-inflammatory and other related molecular expression detection (Western blot or qPCR), to confirm the acclimatization effect.

[0146] VEGF secretion level detection (ELISA): VEGF level ≥800 pg / mL, significantly higher than the normotropic line (50–200 pg / mL).

[0147] Example 2: Preparation method of umbilical cord-derived mesenchymal stem cells (PD-MSCs) based on intermittent gradient hypoxia pretreatment (designated as the intermittent gradient hypoxia group) and cell morphology evaluation

[0148] 1. Isolation and Culture Preparation of Primary MSCs

[0149] 1.1 Umbilical cord source: Fresh umbilical cord tissue from commercially established lines was selected, placed in sterile PBS containing antibiotics (100 IU / mL penicillin, 100 μg / mL streptomycin), and stored at 4°C. The transportation time should not exceed 6 hours.

[0150] 1.2 Primary separation: The tissue was rinsed with sterile PBS in a laminar flow hood, Wharton's jelly was peeled off, and the tissue was cut into 1mm pieces. 3 Tissue blocks. Digested at 37°C for 90 minutes using 0.1% collagenase I / IV (1:1 mixture). After filtering the digestate through a 70 μm filter, cells were collected, centrifuged at 500 × g for 5 minutes, and the supernatant was discarded.

[0151] 1.3 Cell Seeding: Cells were resuspended in serum-free α-MEM complete medium at a concentration of 5,000 cells / cm³. 2 Density seeding was performed in CellSTACK-10 multilayer culture flasks.

[0152] 2. Dynamic hypoxia-noroxic pretreatment

[0153] 2.1 Cultivation Equipment: A programmable low-oxygen incubator was used, with the oxygen program cycle set as follows:

[0154] 5% O2 (24 hours) → 2% O2 (24 hours) → 10% O2 (12 hours) → 21% O2 (12 hours), with each complete cycle lasting 72 hours. This process is repeated for 3-5 rounds of P1-P5 generations, with a total culture time of 9-15 days.

[0155] 2.2 Antioxidant Addition:

[0156] NAC (N-acetylcysteine): Final concentration 1-10 mM

[0157] CoQ10 (Coenzyme Q10): Final concentration 5-20 μM

[0158] 3. Observe cell morphological changes every 24 hours using an inverted microscope, and record cell adhesion, spreading, and density.

[0159] 4. Cell viability detection

[0160] 4.1 After the culture is completed, remove the cells, digest them with 0.25% trypsin, collect them, and freeze them in cryopreservation solution.

[0161] 4.2 Take the frozen and thawed cells, mix the cell suspension with trypan blue staining (0.4% Trypan Blue), load it into an automated cell counter (Countess II FL), count the live cells and dead cells separately, and calculate the overall survival rate.

[0162] The control group, also known as the normoxic group, differs from Example 2 in that the control group was cultured in parallel with the same batch of MSCs in a standard incubator with a constant 21% O2, without the addition of antioxidants.

[0163] Discussion of Results:

[0164] 1. Cell morphology analysis: The PD-MSCs production process in Example 2 includes tissue isolation, cell seeding, dynamic hypoxia-noroxic alternating culture, and the addition of antioxidant factors. The entire process is carried out under sterile and temperature-controlled conditions. The morphology of the harvested MSCs is as follows: Figure 1 As shown, compared with the control group cultured under normoxic conditions, cells cultured under dynamic hypoxia-noroxic alternating conditions maintained good cell morphology and proliferation activity within the same culture cycle.

[0165] 2. Cell viability assessment, such as Figure 2 As shown in the experimental case, the cell viability of MSCs after cryopreservation and thawing under different culture conditions was compared. Multiple experimental results proved that the survival rate of mesenchymal stem cells was significantly improved in the intermittent gradient hypoxia group culture environment. Statistical analysis (Student's t-test) p<0.05, indicating that the difference was statistically significant.

[0166] Example 3: Scratch test of MSC chemotactic migration ability Figure 3 As shown:

[0167] I. Experimental Procedure

[0168] 1. Experimental grouping and cell preparation

[0169] 1.1 The experiment consisted of two groups: the PD-MSC group (the intermittent gradient hypoxia group prepared in Example 2) and the normoxic control group MSC (i.e., the normoxic group).

[0170] 1.2 Two groups of P3 generation cells were collected and their density was adjusted to 1×10⁻⁶ cells using 1% FBS basal medium. 5Cells / mL were seeded into 96-well scratch ablation plates, and the next step was performed after the cell fusion rate reached more than 95%.

[0171] 2. Scratches and Observation

[0172] 2.1 Use a standardized scratching needle to perform mechanical scratching operations to ensure that a scratch of uniform width is formed in the center of each hole.

[0173] 2.2 Gently wash twice with PBS to remove detached dead cells and debris.

[0174] 2.3 The culture plate was photographed under a microscope at 37°C and 5% CO2 for 24 hours.

[0175] II. Experimental Results

[0176] Compared with the normoxic group, the migration ability of PD-MSCs in the intermittent gradient hypoxia group was significantly enhanced. Scratch assays showed that the cell migration rate of PD-MSCs within 24 hours was significantly higher than that in the normoxic group (p<0.05). Figure 3 As shown in the figure. Cell migration rate was quantified using ImageJ software, and the statistical results are as follows. Figure 4 As shown, after 24 hours, the cell migration rate of the control group (i.e., the normoxic group) was about 72%, while the cell migration rate of the PD-MSCs group was about 95%, showing a significant increase in cell migration rate (p<0.001), and the difference was statistically significant.

[0177] Example 4: Real-time quantitative PCR analysis of functional gene expression profiles, as follows Figures 4-7 As shown:

[0178] I. Experimental Procedure

[0179] 1. Experimental grouping and cell treatment

[0180] 1.1 The PD-MSCs obtained from the intermittent gradient hypoxia group in Example 2 and the MSCs cultured in the normoxic group were selected as two control samples.

[0181] 1.2 P5 generation cells were cultured in 6-well plates at a cell density of approximately 8 × 10⁻⁶ cells / well. 4 cells / cm 2 They were cultured in α-MEM + 10% FBS medium at 37°C and 5% CO2 until 80-90% confluence.

[0182] 2. RNA extraction and reverse transcription

[0183] 2.1 Total RNA was extracted using TRIzol reagent, and the purity (OD260 / 280) was considered acceptable at 1.8–2.0.

[0184] 2.2 Using PrimeScript TM Reverse transcription was performed using the RT reagent kit. 1 μg of RNA was added to each reaction tube, with a total reaction volume of 20 μL. The reaction conditions were: 37℃ for 15 min, 85℃ for 5 sec, and stored at 4℃.

[0185] 2.2 Antioxidant addition: NAC (final concentration 5mM) and CoQ10 (final concentration 10μM) were continuously added throughout the culture period.

[0186] 3. Real-time quantitative PCR (qPCR)

[0187] 3.1 The PCR reaction system (20 μL / reaction) includes: Premix Ex Taq II (10 μL), forward and reverse primers (0.4 μL each, 10 μM), cDNA template (2 μL), and ddH2O to bring the total volume to 20 μL.

[0188] 3.2 Functional indicators: including oxygen tolerance (HIF-a), dryness indicators (SOX and OCT4), immunity (IDO), proliferation (Ki67) and migration (HGF).

[0189] II. Experimental Results

[0190] Compared with the control group (noroxic group), the oxygen tolerance level of PD-MSCs in the intermittent gradient hypoxia group was significantly improved, increasing by nearly 5 times (p<0.05). Figure 5 As shown in the figure, it is more conducive to maintaining cell stemness (p<0.05). Both SOX2 and OCT4 indicators showed different levels of improvement, with SOX2 level increasing by 1.3 times (p<0.05) and OCT4 level increasing by 5 times (p<0.05). Figure 6 As shown in the figure, it enhanced immunosuppressive function, and the IDO level was 3 times higher than that of the control group (p<0.05). Figure 7 As shown in the figure), it also promoted cell proliferation, and the Ki67 index increased by 3-4 times (p<0.05). Figure 8 (As shown).

[0191] Example 5: Assessment of VEGF secretion capacity and ROS oxidative stress Figure 9 and Figure 10 As shown:

[0192] I. Experimental Procedure

[0193] 1. VEGF secretion assay (ELISA)

[0194] 1.1 Cell Grouping: PD-MSCs from the intermittent gradient hypoxia group and normoxic cultured MSCs from the normoxic group in Example 2 were selected. P5 cells were seeded in 6-well plates at a density of 1×10⁶ cells per well. 5 Cells were cultured for 48 hours, and the supernatant was collected.

[0195] 1.2 The ELISA kit (R&D Systems, DY293B) was operated according to the instructions. A standard curve was set (concentration range: 31.2-2000 pg / mL), and the absorbance was read at 450 nm.

[0196] 1.3 Each sample group was set up with 3 replicates, and the experiment was repeated 3 times. The average VEGF secretion amount (pg / mL) was calculated.

[0197] 2. ROS content detection (ELISA)

[0198] 2.1 Cell treatment: P5 cells from the same batch were seeded in 6-well plates at a density of 1×10⁶ cells / well. 5 Cells / well, cultured to 80% confluence, then processed.

[0199] 2.2 Staining: Add DCFH-DA working solution and incubate at 37℃ with 5% CO2 for 30 min; wash PBS 3 times to remove excess unabsorbed dye.

[0200] 2.3 Fluorescence Detection

[0201] 96-well plate: Read fluorescence intensity using a multi-mode microplate reader, Ex / Em = 488 / 525nm

[0202] Each group was set up with 3 replicates. The average value was calculated and the blank background was subtracted to obtain the true ROS fluorescence value.

[0203] II. Experimental Results

[0204] 1. VEGF secretion level (e.g.) Figure 9 (As shown)

[0205] The VEGF secretion of PD-MSCs in the intermittent gradient hypoxia group was significantly higher than that in the control group (noroxic group), increasing by about 5-6 times (p<0.01).

[0206] 2. ROS oxidation level (e.g.) Figure 10 (As shown)

[0207] Example data from CCK-8 cell viability assay (three biological replicates, OD measurement in 96-well plates) 450 Experimental design: Cells: MSC culture conditions: normoxic (21% O2) vs hypoxic (2-10% O2), treatment: ±200 μM H2O2 shock for 2 h, followed by detection after 24 h recovery.

[0208] Figure 9 and Figure 10 The differences in VEGF secretion capacity and oxidative stress levels between the two groups of cells were demonstrated.

[0209] Conclusion: PD-MSCs exhibited stronger pro-angiogenic factor release capacity and significantly reduced ROS levels under alternating hypoxia-noroxic stimulation and antioxidant treatment, indicating enhanced antioxidant capacity.

[0210] Example 6: Operational procedures for drug delivery routes at different sites (e.g.) Figure 11 (As shown)

[0211] I. Experimental Objective

[0212] This embodiment 6 aims to systematically establish and standardize the operation procedures of three MSC administration routes in a mouse model of premature ovarian failure, forming a unified preclinical evaluation reference model, highlighting the technical highlights of this invention in "innovative drug administration strategies".

[0213] II. Operation Flow Design

[0214] Table 1: Three Mesenchymal Stem Cell Infusion Routes

[0215]

[0216]

[0217] Three path operation and evaluation flowcharts: Figure 7 The key steps of each pathway are shown in flowchart form, including: animal anesthesia → site localization → infusion method → ​​infusion dose and volume → postoperative observation → efficacy evaluation.

[0218] The core nodes of the process are as follows:

[0219] 1. Animal preparation

[0220] a) C57BL / 6 female mice, 6–8 weeks old.

[0221] b) Modeling was performed by intraperitoneal injection of CTX 50mg / kg for 15 days.

[0222] c) Fasting for 6 hours before administration, and isoflurane inhalation anesthesia.

[0223] 2. Injection pathway procedure:

[0224] a) Route A: Open the abdominal cavity, expose the ovary, and inject 200 μL containing 2×10⁻⁶ ozonates subcapsularly. 6 PD-MSCs suspension, microscopic-assisted positioning, and wound closure.

[0225] b) Route B: Inject 200 μL of cell solution into the tail root vein using a 30G injection needle. Observe for the absence of reflux and nodules to indicate a qualified injection.

[0226] c) Pathway C: Ultrasound-guided ovarian artery, microcatheter puncture and injection, avoiding excessive pressure and vascular rupture.

[0227] 3. Postoperative management:

[0228] Mice were placed on a warming pad to recover, and their mental state and the injection site were monitored for 24 hours without any inflammatory redness or swelling.

[0229] IV. Experimental Procedure

[0230] 1. Serum hormone testing on day 49

[0231] 1.1 Blood Collection and Serum Preparation

[0232] On the morning of day 49 after drug administration, mice in each group were anesthetized with sodium pentobarbital (50 mg / kg, ip) and 1.5–2 mL of blood was collected by cardiac puncture. After standing for 30 min to allow the blood to clot, the blood was centrifuged at 3000g for 10 min at 4℃, and the supernatant serum was collected and stored at -80℃ for later use.

[0233] 1.2 ELISA assay

[0234] The R&D Systems mouse-specific FSH enzyme-linked immunosorbent assay kit was used. All serum samples were diluted 1:5 with PBS and operated according to the instructions. The optical density was read at 450 nm, and a standard curve was generated using four-parameter nonlinear regression to calculate the concentration.

[0235] 1.3 Statistical Methods

[0236] Using GraphPadPrism9 software, the Shapiro–Wilk normality test was first performed; after confirming that the data conformed to a normal distribution, a one-way ANOVA was used, and the Dunnett test was used for post-hoc comparisons, with a significance level of α = 0.05.

[0237] 2. Assessment of breeding in cages starting from day 60

[0238] 2.1 Mating and Observation

[0239] Sixty days after the end of drug administration, female mice in each group were housed with healthy 12-week-old male mice at a 1:1 ratio and observed for 30 consecutive days. The vaginal opening was checked daily in the morning for plugging to confirm mating, and the time of delivery and the number of newborn pups were recorded.

[0240] 2.2 Statistical Indicators

[0241] Pregnancy rate (%) = (Number of female mice giving birth / Total number of female mice in the group) × 100

[0242] Average litter size = Sum of all litters in the group / Number of females giving birth

[0243] 2.3 Topographical Photography

[0244] a) After 30 days of mating and birthing, place the birthing cage on a biosafety workbench on the day the pups are born (P0).

[0245] b) Close the cage lid, keep the original bedding and nesting material in place, and remove the water bottle and food bowl without disturbing the mother mouse;

[0246] c) Use the same digital SLR camera (Canon EOS 90D, lens 18–55mm) fixed on a tripod, with the lens vertically aligned with the center of the cage top net, 35cm away from the surface of the padding material, aperture F=8, shutter speed 1 / 125s, ISO400; use a ring LED fill light to ensure uniform illumination.

[0247] d) Take three top-view photos of each mother mouse that has given birth, and select the photo in which all the pups are clearly visible as the counting photo; if the mother mouse blocks the view, gently tap the cage wall to guide her to leave the nest temporarily and then quickly complete the photo (lasting <3 seconds).

[0248] V. Experimental Results

[0249] 1. In this case study, the therapeutic and repair effects of three drug delivery routes—ovarian capsule, tail vein, and ovarian artery—were compared. The groups were as follows: Figure 11 As shown.

[0250] 2. Estradiol valerate was used as a positive control group. Samples were collected and taken on day 49 to evaluate hormone levels. Figure 12 ), and fertility ( Figure 13 ) and morphological diagrams of mouse pups ( Figure 14 A comprehensive evaluation of the therapeutic effect of MSC on premature ovarian failure.

[0251] 2.1 Comparison of FSH hormone levels in different groups of mice

[0252] Follicle-stimulating hormone (FSH) is secreted by the pituitary gland and is used to promote follicle development. Figure 12 It is evident that the POF group exhibited typical elevated FSH levels, consistent with the typical characteristics of POF. After intervention, FSH levels in all treatment groups decreased significantly (P<0.01). Under the same administration route, the PD-MSCs group was more effective than the NR-MSCs group (p<0.05). Among the three administration routes, Oi and Ti were slightly more effective than Oai.

[0253] 2.2 Comparison of fertility in different groups of mice

[0254] Comparing the reproductive rates of mice in different groups revealed ( Figure 13 The pregnancy rate in the PD-MSCs group (Oi injection group) was 83%, which was superior to that in the NR-MSCs group and the positive drug group (p<0.05).

[0255] 2.3. Comparison of morphological images of offspring born to mice in different groups

[0256] Comparison of the number of offspring and mouse morphology in different groups revealed no significant differences in weight, shape, or fur between the mice born in each treatment group and the blank control group. Figure 14 The results showed that the PD-MSCs+Oi group had a total of 25 offspring, which was better than the NR-MSCs group and the positive drug group (p<0.05). Combined with the comparison chart of mouse fertility, it is suggested that the PD-MSCs+Oi treatment method has the best effect on restoring fertility.

[0257] Animal treatment efficacy evaluation

[0258] Experimental animals: Female C57BL / 6 mice, 6–8 weeks old, induced with CTX for 15 consecutive days to induce premature ovarian failure;

[0259] Treatment method: A single infusion of PD-MSCs cell suspension (2×10^6 cells / animal) on day 21;

[0260] Comparison of routes of administration (innovative evaluation of efficacy via multiple routes): tail vein administration; subcapsular injection of the ovary; ovarian artery injection;

[0261] The control group was set up with estradiol valerate (positive control) to ensure the reliability of the model evaluation.

[0262] Multidimensional evaluation of the endpoint on day 49: CTX-induced POF model, combined with serum hormone (FSH), reproductive rate and photographs of fertile individuals.

[0263] Table 2: Changes in the characteristics of mesenchymal stem cells

[0264]

[0265] The PD-MSCs obtained by alternating hypoxia treatment in this application are specifically designed for the treatment of CTX-induced ovarian failure (POF) models, rather than being limited to musculoskeletal or osteoarthritis applications. Their uses encompass multiple therapeutic effects, including repairing ovarian tissue structure, improving fertility outcomes, and restoring endocrine function. This application provides a complete integrated solution of "hypoxia treatment + culture system + passage process + combined delivery + indication matching + indicator verification," emphasizing the synergistic optimization and system construction capabilities between different technical links to form a systemic technical barrier.

[0266] The key design focus of this invention is that, through the intermittent gradient hypoxia pretreatment-based mesenchymal stem cells, their preparation method, and their application in drugs for treating premature ovarian failure, this application effectively addresses the shortcomings of existing technologies in MSC preparation and administration, specifically manifested in the following prominent advantages:

[0267] 1. Dynamic hypoxia pretreatment significantly improves the ability of MSCs to adapt to the special pathological environment of the ovary:

[0268] The dynamic, intermittent gradient hypoxia-noroxic alternating culture method with 2%–10% O2 concentration effectively simulated the pathological characteristics of "hypoxia-reoxygenation" in ovarian tissue after chemotherapy, activated the HIF-1α signaling pathway and related antioxidant and anti-apoptotic mechanisms, and significantly improved the survival rate and functional activity of MSCs in the ovary.

[0269] 2. Shorten the domestication cycle to maintain the youthfulness and functionality of MSC cells:

[0270] Limiting the MSC domestication process to the P2–P5 generation avoids cell senescence and telomere shortening caused by long-term expansion, significantly improves the paracrine capacity and repair efficiency of MSCs, and ensures the high efficiency and stable biological activity of MSCs.

[0271] 3. Introducing antioxidants significantly reduces oxidative damage to MSCs:

[0272] The addition of N-acetylcysteine ​​(NAC) and coenzyme Q10 (CoQ10) as antioxidants during the culture process effectively alleviates oxidative stress generated during the alternating hypoxia-reoxygenation culture process, significantly reduces cell damage, and maintains the stability and high efficiency of cell therapy preparations.

[0273] 4. Combining multiple drug delivery routes to optimize MSC homing and repair efficiency:

[0274] This study innovatively compared three different MSC infusion routes: subcapsular injection, tail vein injection, and ovarian artery injection. The results showed that, through comprehensive evaluation of multiple indicators, including serum hormone levels, follicle count in HE-sectioned ovarian tissue, observation of fertility outcomes, and expression of molecular markers (such as pro-angiogenic factors and anti-inflammatory factors), the combined administration strategy significantly improved the actual therapeutic effect of MSCs and the ovarian repair capacity.

[0275] 5. The evaluation indicators are multi-dimensional and multi-level, objectively and comprehensively verifying the efficacy of MSC:

[0276] In the animal model evaluation, a mouse model of premature ovarian failure was induced by CTX for 15 consecutive days (highly simulating the clinical chemotherapy scenario); on day 21, a single infusion of 2×10^6 PD-MSCs was performed, and a positive control (estradiol valerate) was established to ensure the effectiveness of the model; on day 49, a comprehensive evaluation was carried out on serum hormones (FSH, E2, AMH, etc.), follicle count in HE slides, fertility assessment (pregnancy rate, number of fetuses), and various molecular indicators (VEGF, HGF, TNF-α, IL-6, etc.) to achieve a multidimensional and comprehensive evaluation of cell and animal models, effectively overcoming the misjudgment of efficacy caused by single or one-sided evaluation indicators in the past, and enhancing the objectivity and credibility of the efficacy.

[0277] 6. A functional quality control indicator system to ensure MSC batch stability:

[0278] A multifunctional indicator detection system was introduced, including cell phenotype (CD73, CD90, CD105 expression), oxygen stress pathway molecules (HIF-1α, CXCR4), and cell metabolism indicators (OCR / ECAR ratio), which ensured a high degree of consistency in the quality and therapeutic function of the prepared MSCs between batches, effectively solving the problems of the traditional MSC quality evaluation system being weak and unstable.

[0279] 7. Significantly improves overall ovarian function and fertility recovery:

[0280] Animal models have validated that the MSC treatment group of this patent is significantly superior to single-pathway drug administration or conventional hypoxic / noroxic culture MSC treatment protocols in multiple indicators, including endocrine hormone regulation, follicle structure recovery, improved fertility, reduced inflammatory factors, and ovarian angiogenesis. This indicates that the innovative protocol not only achieves a technological breakthrough in cell preparation but also has significant advantages in therapeutic effects, demonstrating the technological advancement and clinical application prospects of the dual dimensions of cell and animal evaluation.

[0281] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing mesenchymal stem cells based on intermittent gradient hypoxia pretreatment, characterized in that: Includes the following steps: S1. Isolation of primary mesenchymal stem cells and inoculation in serum-free α-MEM complete medium; S2. Primary mesenchymal stem cells were cultured in serum-free α-MEM complete medium under intermittent gradient hypoxia-noroxic alternating pretreatment. S3. Cell passage and expansion: Harvest passaged mesenchymal stem cells.

2. The method for preparing mesenchymal stem cells based on intermittent gradient hypoxia pretreatment according to claim 1, characterized in that: The intermittent gradient hypoxia-noroxic alternating pretreatment culture in step S2 is specifically as follows: On the first day, the cells were cultured for 24 hours under normal aerobic conditions: 21% O2, 5% CO2, and 37°C to activate initial proliferation. Starting the next day, an intermittent alternation method was used for oxygen tension switching culture, namely: hypoxia condition: oxygen concentration of 2%–10% O2; normoxic condition: oxygen concentration of 21% O2; the hypoxia condition and normoxic condition were alternated every 12–36 hours, and the cycle was repeated 6–12 times.

3. The method for preparing mesenchymal stem cells based on intermittent gradient hypoxia pretreatment according to claim 1, characterized in that: The serum-free α-MEM complete culture medium in step S1 includes: α-MEM basal culture medium: 90%–95% by volume; human platelet lysate (PLTGold): 2%–7% by volume; nutrient additive (Nutri-Add): 0.5%–3% by volume and antioxidant.

4. The method for preparing mesenchymal stem cells based on intermittent gradient hypoxia pretreatment according to claim 3, characterized in that: In step S1, the antioxidants are selected from N-acetylcysteine ​​(NAC) with a final concentration of 1-5 mM; glutathione (GSH) with a final concentration of 0.1-1 mM; and vitamin C with a final concentration of 100-200 μM. Lipoic acid: final concentration 50–100 μM and coenzyme Q10: final concentration 2–20 μM.

5. The method for preparing mesenchymal stem cells based on intermittent gradient hypoxia pretreatment according to claim 4, characterized in that: The antioxidants in step S1 include N-acetylcysteine ​​(NAC) at a final concentration of 1-5 mM and coenzyme Q10 (CoQ10) at a final concentration of 2-20 μM.

6. The method for preparing mesenchymal stem cells based on intermittent gradient hypoxia pretreatment according to claim 1, characterized in that: The cell passage and expansion in step S3 specifically includes the following steps: S31. When the cell confluence reaches 80%, digest with 0.25%-0.5% trypsin / EDTA, and then neutralize with 3-5 times the volume of serum-free α-MEM complete medium. Centrifuge at 300g-500g for 5-10 minutes, discard the supernatant, and resuspend the cells; S33. Passage the cells at a ratio of 1:3–1:4 and continue intermittent alternating hypoxia-noroxic culture, i.e.: hypoxia condition: oxygen concentration of 2%–10% O2; normoxic condition: oxygen concentration of 21% O2; alternate between hypoxia and normoxic conditions every 12–36 hours, for 6–12 cycles; harvest P4 or P5 generation mesenchymal stem cells.

7. The method for preparing mesenchymal stem cells based on intermittent gradient hypoxia pretreatment according to claim 1, characterized in that: Step S1 specifically includes the following steps: S11. Selection of primary mesenchymal stem cell sources and isolation of primary mesenchymal stem cells; S12. Cell Seeding: Primary mesenchymal stem cells were resuspended in serum-free α-MEM complete medium and seeded at 5,000 cells / cm³. 2 Density seeding was carried out in the culture platform.

8. A mesenchymal stem cell prepared using the method for preparing mesenchymal stem cells based on intermittent gradient hypoxia pretreatment as described in any one of claims 1-7.

9. The application of mesenchymal stem cells as described in claim 8 in a drug for treating premature ovarian failure, characterized in that: The mesenchymal stem cells are prepared as a drug for treating premature ovarian failure.

10. The application according to claim 9, characterized in that: The administration method of the drug is selected from any one of subcapsular injection of the ovary, tail vein injection, and ovarian artery injection.

Images