Menstrual blood-derived mesenchymal stem cell ovarian targeting complex and preparation method thereof

Abstract

The application provides a blood-derived mesenchymal stem cell ovary targeting complex and a preparation method thereof. The ternary gel matrix system of the application is composed of sodium hyaluronate, four-arm polyethylene glycol maleimide and mercapto carboxymethyl dextran in the second precursor solution, a thioether bond crosslinking network is formed in situ through a maleimide-mercapto Michael addition reaction, and ovarian decellularized extracellular matrix microfilaments are introduced as a biomimetic support unit, and the blood-derived mesenchymal stem cells are encapsulated into injectable microtissue unit preparations with an arithmetic mean of 150-800 mu m and a particle size variation coefficient of not more than 15% through a microfluidic device. The application realizes the synergistic optimization of the injectable fluidity of the gel matrix and the rapid forming stability in the body, significantly improves the cell viability protection effect and the microtissue unit batch particle size uniformity, and solves the technical contradiction that the injectability and structural stability cannot be considered in the existing injectable cell preparations.

Description

Technical Field

[0001] This invention relates to the fields of cell therapy and biomaterials technology, specifically to a menstrual blood-derived mesenchymal stem cell ovarian-targeting complex and its preparation method. Background Technology

[0002] The ovary is a vital reproductive endocrine organ in women. Early-onset ovarian insufficiency caused by ovarian dysfunction is a significant cause of infertility and endocrine disorders in women of reproductive age, with an incidence exceeding 1% and showing a trend towards affecting younger women. This poses a serious threat to patients' physical and mental health and quality of life. Against this backdrop, in situ targeted therapy utilizing the paracrine regulation, anti-apoptosis, and immunomodulatory properties of mesenchymal stem cells (MSCs) is considered a highly promising treatment approach for repairing damaged ovarian tissue, promoting follicle development, and restoring endocrine function. Menstrual blood-derived MSCs, as an important subpopulation of exfoliated endometrial cells, possess unique advantages such as non-invasive acquisition, abundant source, strong proliferative capacity, and low immunogenicity. Furthermore, due to their inherent homing imprint of the endometrial microenvironment, they exhibit significant tissue compatibility and functional adaptability in the field of ovarian endocrine repair. In situ ovarian injection is the core pathway for achieving local targeted delivery of stem cells. However, constructing a carrier formulation system that combines injectable fluidity, rapid in situ in vivo prototyping, and cell viability protection is a key technological bottleneck in moving this therapy from the laboratory to clinical application. To meet the engineering constraints of transvaginal ultrasound-guided injection with long-tube puncture needles, and to ensure the spatial retention stability of cells and the integration function of the ovarian microenvironment after injection, a systematic and synergistic design of the rheological properties, gel dynamics, and biocompatibility of the formulation system is required. This poses multi-dimensional and complex requirements for the development of high-performance injectable carrier materials.

[0003] Currently, research on injectable hydrogel systems for in situ injection of stem cells into the ovary has made some progress, but existing technologies still have significant limitations in meeting multiple performance requirements. Most existing systems use single-component natural polysaccharides (such as hyaluronic acid, gelatin, and alginate) or synthetic polymers (such as polyethylene glycol) to form the gel matrix. Such approaches often present an inherent contradiction between gelation rate and mechanical stability: insufficient cross-linking density results in a loose microstructure, uneven cell distribution, and poor in vivo retention after injection; excessively high cross-linking density leads to a significant increase in gel viscosity, causing significant shear damage to cells during injection and obstructing mass transfer channels between cells and the matrix, further affecting cell function. Furthermore, existing systems generally lack biomimetic support components corresponding to the natural extracellular matrix of the ovary, resulting in a lack of specific biological signal interactions between cells and the matrix, limiting the maintenance of stem cell function and integration into ovarian tissue after implantation. Moreover, existing injectable microspheres or microgel systems are mostly prepared using batch emulsification methods, resulting in wide particle size distributions and large batch-to-batch variability, making it difficult to meet the requirements of uniformity and reproducibility for clinical administration. For example, Chinese patent application CN119215063B discloses a modified hyaluronic acid injectable formulation for inflammation targeting and stem cell capture, its preparation method, and its uses. However, this formulation uses a single-component gel matrix, lacks biomimetic extracellular matrix microstructure support, and has insufficient means to control particle size uniformity, resulting in the risk of cell viability damage and poor batch-to-batch stability. Therefore, developing novel injectable micro-tissue unit formulations that can simultaneously address the synergistic needs of injectability, rapid prototyping, cell viability protection, biomimetic support, and particle size uniformity has significant technological value and clinical implications. Summary of the Invention

[0004] The purpose of this invention is to provide a blood-derived mesenchymal stem cell ovarian-targeting complex and its preparation method, which solves multiple technical contradictions in current injectable cell / hydrogel micro-tissue systems, such as the difficulty in balancing injectability and in vivo molding stability, the mutual constraint between micro-tissue unit particle size uniformity and cell viability maintenance, and the decrease in manufacturability caused by the introduction of ovarian biomimetic components.

[0005] This invention comprehensively integrates the lubricating and flow contribution of sodium hyaluronate, the controllable cross-linking kinetics of the maleimide-mercaptoMichael addition reaction between the four-arm polyethylene glycol maleimide and the thiolated carboxymethyl dextran in the second precursor solution, and the biomimetic fiber toughening effect of ovarian decellularized extracellular matrix microfilaments, achieving synergistic effects through precise formulation of the three components. Sodium hyaluronate, through physical embedding, imparts hydrophilic lubricity and cellular responsiveness to the gel matrix, significantly reducing injection resistance; the in-situ cross-linking of thioether bonds between the four-arm polyethylene glycol maleimide and the thiolated carboxymethyl dextran in the second precursor solution provides rapid in vivo molding capability and structural stability; the ovarian decellularized extracellular matrix microfilaments provide biomimetic support for the fibrous skeleton, compensating for the deficiency of single biological signals in synthetic networks; and hypoxia pretreatment further activates the paracrine potential of menstrual blood-derived mesenchymal stem cells. The above four-dimensional synergistic effect enables a qualitative leap in overall performance, and multiple functions that cannot be achieved by individual components are integrated and co-presented in this system.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A menstrual blood-derived mesenchymal stem cell ovarian-targeting complex, the complex comprising an injectable microtissue unit formulation, the injectable microtissue unit formulation being composed of multiple microtissue units, the arithmetic mean of the equivalent particle size of the microtissue units being 150-800 μm, and the coefficient of variation of the equivalent particle size being no higher than 15%; The equivalent particle size is the equivalent diameter of the micro-tissue unit calculated based on the projected area in a microscopic image; the injectable micro-tissue unit formulation includes blood-derived mesenchymal stem cells, ovarian acellular extracellular matrix microfilaments, and a gel matrix; the gel matrix contains sodium hyaluronate, tetra-arm polyethylene glycol maleimide, and thiolated carboxymethyl dextran in the second precursor solution. The four-armed polyethylene glycol maleimide and the thiolated carboxymethyl dextran in the second precursor solution form a thioether cross-linked structure through a maleimide-thioMichael addition reaction.

[0007] Furthermore, the thiolated carboxymethyl dextran in the second precursor solution is prepared via the following steps: A1. Mix sodium carboxymethyl dextran, β-mercaptoethylamine, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; A2. Under conditions of pH 4.5-6.0, activate the carboxyl group of sodium carboxymethyl dextran and react it with β-mercaptoethylamine for 2-24 h; A3. The reaction is terminated when the thiol content of the obtained polymer, on a dry basis, is 0.05-0.50 mmol / g; A4. Unreacted small molecules were removed by dialysis, and the thiolated carboxymethyl dextran was obtained after drying.

[0008] Furthermore, the ovarian decellularized extracellular matrix microfilaments are prepared through the following steps: B1. Cut the pig ovarian tissue and wash it with sodium chloride solution; B2. Decellularization treatment: The porcine ovarian tissue was treated in an aqueous solution of sodium dodecyl sulfate with a mass-volume percentage concentration of 0.1-1.0% (w / v), then treated in a Triton X-100 aqueous solution, and then chelated and washed with an aqueous solution containing disodium ethylenediaminetetraacetate dihydrate. B3. Nucleic acid removal: The tissue treated in step B2 is placed in an aqueous solution containing deoxyribonuclease I and anhydrous magnesium chloride, wherein the anhydrous magnesium chloride is used to provide magnesium ions; B4. Endpoint criteria: The obtained decellularized extracellular matrix meets the following requirements: double-stranded deoxyribonucleic acid content not exceeding 50 ng / mg dry weight, deoxyribonucleic acid fragment length not exceeding 200 base pairs, and no visible cell nuclei in the tissue sections; B5. Microfilamentization: The decellularized extracellular matrix is ​​sheared or ground to obtain ovarian decellularized extracellular matrix microfilaments with a length of 50-150 μm and a diameter of 1-30 μm.

[0009] Furthermore, the menstrual blood-derived mesenchymal stem cells are derived from human menstrual blood and are CD73-positive, CD90-positive, CD105-positive, CD34-negative, and CD45-negative; the menstrual blood-derived mesenchymal stem cells are pretreated with hypoxia before encapsulation.

[0010] Furthermore, the injectable microtissue unit formulation is prepared using a microfluidic device, and the coefficient of variation of the equivalent particle size of the microtissue unit is not higher than 15%.

[0011] Furthermore, the mass-volume percentage concentration of sodium hyaluronate in the first precursor solution is 0.5-2.0% (w / v), the mass-volume percentage concentration of tetra-arm polyethylene glycol maleimide is 3.0-7.0% (w / v), and the mass-volume percentage concentration of thiolated carboxymethyl dextran in the second precursor solution is 0.1-2.0% (w / v).

[0012] Furthermore, the molecular weight of the four-arm polyethylene glycol maleimide is 10,000-40,000 Da, and the substitution rate of the terminal maleimide group is not less than 90%.

[0013] Furthermore, the content of the ovarian decellularized extracellular matrix microfilaments in the injectable microtissue unit formulation is 0.1-30 mg / mL.

[0014] As a concept of the present invention, the present invention adopts a design that combines maleimide-thiol Michael addition crosslinking with biomimetic enhancement of ovarian decellularized extracellular matrix microfilaments, which is mainly used to enhance the comprehensive performance and in vivo functional expression of injectable microtissue unit formulations.

[0015] The gel matrix system of this invention uses the maleimide-thiol Michael addition reaction as the core crosslinking chemistry. This reaction, under near-neutral physiological pH conditions, rapidly and quantitatively forms stable thioether bonds between the terminal maleimide groups of the four-arm polyethylene glycol maleimide and the thiol groups of the thiolized carboxymethyl dextran side chains in the second precursor solution. The crosslinking reaction rate can be precisely controlled by component concentration and pH, thereby limiting the gelation time to the 1-15 min window required for microfluidic molding, achieving temporal decoupling between the injectable liquid dispersion and rapid in vivo solidification. Sodium hyaluronate exists physically embedded in the thioether bond crosslinking network, without participating in chemical crosslinking. This imparts excellent hydrophilic lubricity to the matrix to reduce injection shear resistance, and through its inherent CD44 receptor ligand effect, provides adhesion and survival signals for blood-derived mesenchymal stem cells, while preserving the network's cellular remodeling window. The four-armed polyethylene glycol maleimide's four-functional star-shaped topology endows the cross-linked network with a highly homogeneous pore size distribution, ensuring mass transfer while maintaining the micro-tissue unit's anti-fracture stability. In the second precursor solution, the thiolated carboxymethyl dextran backbone is rich in hydroxyl and carboxyl groups, and its hydrophilicity and biocompatibility further reduce the non-specific adsorption and toxicity of the gel matrix to encapsulated cells. Ovarian decellularized extracellular matrix microfilaments, as biomimetic fiber units, retain natural matrix components such as collagen, glycosaminoglycans, and growth factors, which can directly activate the ovarian-targeted paracrine program of blood-derived mesenchymal stem cells and form a fibrous skeleton within the micro-tissue unit, significantly improving the unit's mechanical toughness against needle shear.

[0016] This invention also discloses a method for preparing a menstrual blood-derived mesenchymal stem cell ovarian-targeting complex, wherein the complex is an injectable micro-tissue unit formulation, comprising the following steps: S1, providing thiolated carboxymethyl dextran and ovarian decellularized extracellular matrix microfilaments in the second precursor solution; S2. Preparation of gel matrix precursor: Prepare a first precursor solution containing sodium hyaluronate and tetra-armed polyethylene glycol maleimide and a second precursor solution containing thiolated carboxymethyl dextran from the second precursor solution. The first precursor solution and the second precursor solution are stored separately before step S3. S3. Combining and Unitization: Menstrual blood-derived mesenchymal stem cells and ovarian decellularized extracellular matrix microfilaments are added to the second precursor solution and mixed. The first precursor solution and the second precursor solution containing menstrual blood-derived mesenchymal stem cells and ovarian decellularized extracellular matrix microfilaments are contacted and mixed through a microfluidic device to form micro-tissue units. The four-armed polyethylene glycol maleimide undergoes a maleimide-mercapto-Michael addition reaction with the thiolated carboxymethyl dextran in the second precursor solution. The crosslinking time is 1-15 min from the time the first precursor solution and the second precursor solution come into contact and mix in the microfluidic device, resulting in an injectable micro-tissue unit formulation.

[0017] Furthermore, in step S2, the mass-volume percentage concentration of sodium hyaluronate in the first precursor solution is 0.5-2.0% (w / v), and the mass-volume percentage concentration of tetra-arm polyethylene glycol maleimide is 3.0-7.0% (w / v); the mass-volume percentage concentration of thiolated carboxymethyl dextran in the second precursor solution is 0.1-2.0% (w / v).

[0018] As another aspect of this invention, a preparation process design employing stepwise preparation of dual precursor solutions and precise microfluidic shaping is employed. This is primarily used to enhance the particle size uniformity, batch-to-batch reproducibility, and aseptic controllability of injectable microtissue unit formulations. In this invention, the first precursor solution containing maleimide groups and the second precursor solution containing thiol groups are strictly stored separately before step S3. The fundamental reason is that the maleimide-thiol Michael addition reaction initiates rapidly after mixing. This separation strategy effectively extends the shelf life of each precursor solution, ensuring the activity and precise proportioning of components within the preparation window. At the instant the two liquids come into contact and mix within the microfluidic device channel, the cross-linking reaction is precisely triggered. Combining the principles of microfluidic flow focusing or co-flow focusing, the dispersed phase droplets form highly uniform microdroplet units (coefficient of variation ≤15%) within the channel, completing gelation and solidification. This ensures a high degree of consistency between the number of blood-derived mesenchymal stem cells encapsulated in each microtissue unit and the distribution of ovarian decellularized extracellular matrix microfilaments. Precise control of crosslinking time (1-15 min) provides ample operating window for the process while avoiding network shrinkage and mass transfer barriers caused by over-crosslinking. Pre-dispersing menstrual blood-derived mesenchymal stem cells in the second precursor solution, rather than adding them during mixing, effectively avoids mechanical shear damage to the cell membrane caused by high-speed mixing. Combined with hypoxia pretreatment to pre-activate the paracrine program of menstrual blood-derived mesenchymal stem cells, the functional state of cells is optimized before formulation, thereby synergistically achieving cell viability protection and precise micro-tissue unit formation.

[0019] Furthermore, the present invention also discloses a kit comprising: a) an injectable microtissue unit formulation; and b) a long-tube puncture injection assembly.

[0020] Furthermore, the arithmetic mean of the equivalent particle size of the micro-organism is 150-800 μm, and the coefficient of variation of the equivalent particle size is not higher than 15% among the equivalent particle sizes measured by randomly selecting no less than 200 micro-organisms in the same batch of formulation.

[0021] Furthermore, the equivalent particle size is the area equivalent diameter d corresponding to the projected area A of the microstructure unit in the microscopic image, where d = (4A / π). 0.5 .

[0022] Furthermore, in the gel matrix, the molar ratio of maleimide groups to thiol groups is 0.8:1-1.2:1, calculated based on the maleimide groups provided by the four-arm polyethylene glycol maleimide and the thiol groups provided by the thiolized carboxymethyl dextran in the second precursor solution. The number of moles of maleimide groups is calculated based on the feed mass, molecular weight, four-arm functionality, and substitution rate of the terminal maleimide groups of the four-arm polyethylene glycol maleimide. The number of moles of thiol groups is calculated based on the feed mass of the thiolized carboxymethyl dextran in the second precursor solution and the thiol content on a dry basis.

[0023] Furthermore, the sodium hyaluronate exists in a physically embedded manner within the thioether crosslinking network formed by the four-arm polyethylene glycol maleimide and the thiolated carboxymethyl dextran in the second precursor solution.

[0024] Furthermore, in step S2, the first precursor solution is prepared by dissolving sodium hyaluronate and tetra-arm polyethylene glycol maleimide in deionized water or phosphate buffer at pH 6.5-7.5, and the second precursor solution is prepared by dissolving the thiolated carboxymethyl dextran in deionized water or phosphate buffer at pH 6.5-7.5.

[0025] Furthermore, the first precursor solution is sealed and stored at 4°C after preparation, and used in step S3 within 12 hours after preparation; the second precursor solution is sealed and stored at 4°C after preparation, and used in step S3 within 24 hours after preparation.

[0026] Further, in step S3, under aseptic conditions, mesenchymal stem cells derived from menstrual blood and ovarian decellularized extracellular matrix microfilaments are added to the second precursor solution and mixed to obtain a dispersed aqueous phase. The first precursor solution and the dispersed aqueous phase are then contacted and mixed in a microfluidic device to form micro-tissue units. The crosslinking time is 1-15 min from the time the first precursor solution and the dispersed aqueous phase come into contact and mix in the microfluidic device. After crosslinking is completed, the obtained micro-tissue units are recovered from the continuous phase and washed at least 3 times with phosphate buffer until no visible oil droplets are found in the supernatant after washing and no oil phase separation occurs after standing for 10 min.

[0027] Further, in step A1, sodium carboxymethyl dextran with a weight-average molecular weight of 10,000-500,000 Da and a degree of substitution of 0.6-1.2 is dissolved in MES buffer or phosphate buffer with a molar concentration of 0.05-0.20 mol / L and the pH is adjusted to 5.0-6.0 to obtain a sodium carboxymethyl dextran solution with a mass-volume percentage concentration of 0.5-3.0% (w / v). Under continuous stirring, β-mercaptoethylamine, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added to the solution, wherein the mass ratio of sodium carboxymethyl dextran to β-mercaptoethylamine is 1:0.02-0.30, and the molar ratio of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 0.8-2.0:1.

[0028] Furthermore, in step A2, the reaction is carried out at 20-40°C for 2-24 h in MES buffer or phosphate buffer with a molar concentration of 0.05-0.20 mol / L, pH 4.5-6.0, and under continuous stirring.

[0029] Furthermore, in step A3, when the thiol content of the obtained polymer, measured by the Ellman reagent method or iodometric titration, is 0.05-0.50 mmol / g on a dry basis, stirring is stopped and the dialysis post-treatment in step A4 is immediately initiated to terminate the coupling reaction.

[0030] Furthermore, in step A4, a dialysis bag with a molecular weight cutoff of 3500-14000 Da is used, and dialysis is performed in deionized water or a buffer solution with a pH of 6.0-8.0 for 24-72 hours, with the dialysis solution being replaced every 8-12 hours.

[0031] Furthermore, in step A4, after dialysis, the thiolated carboxymethyl dextran in the second precursor solution is obtained by freeze-drying or vacuum drying. The freeze-drying is performed by freezing at -40 to -80°C and then drying at a vacuum degree not exceeding 50 Pa and a temperature not exceeding -20°C for 24-72 h. The vacuum drying is performed by drying at a vacuum degree not exceeding 100 Pa and a temperature of 40-60°C for 12-48 h.

[0032] Furthermore, in step B1, the pig ovarian tissue is cut into pieces and washed 2-5 times with a sodium chloride aqueous solution with a mass-volume percentage concentration of 0.9% (w / v), with each washing solution volume being 5-20 times the tissue mass, and the washing temperature being 4-25℃.

[0033] Furthermore, in step B2, the porcine ovarian tissue is placed in an aqueous solution of sodium dodecyl sulfate with a mass-volume percentage concentration of 0.1-1.0% (w / v), and the ratio of liquid volume to tissue mass is 10-50 mL / g. The tissue is then treated on a shaker at 50-150 rpm for 4-48 h at room temperature to 37°C.

[0034] Furthermore, in step B2, the tissue is then placed in a Triton X-100 aqueous solution with a volume fraction of 0.5-2.0 vol%, the volume-to-tissue ratio being 10-50 mL / g, and treated on a shaker at 50-150 rpm for 2-24 h at room temperature to 37°C.

[0035] Furthermore, in step B2, the chelation washing is performed using an aqueous solution of disodium ethylenediaminetetraacetate dihydrate with a mass-volume percentage concentration of 0.037-0.744% (w / v) corresponding to a molar concentration of 1-20 mmol / L. The pH value is 7.0-8.0, the washing is performed 2-5 times, and the washing time is 10-60 min each time.

[0036] Furthermore, in step B3, the concentration of deoxyribonuclease I is 10-200 U / mL, the concentration of anhydrous magnesium chloride is 1-10 mmol / L corresponding to a mass-volume percentage concentration of 0.0095-0.095% (w / v), the pH of the aqueous solution is 7.5-8.0, the treatment temperature is 25-37℃, the treatment time is 1-6 h, and the ratio of liquid volume to tissue mass is 10-50 mL / g.

[0037] Furthermore, in step B4, the content of double-stranded deoxyribonucleic acid is determined by nucleic acid quantification, and the length of the deoxyribonucleic acid fragment is determined by gel electrophoresis or equivalent fragment length analysis.

[0038] Furthermore, in step B5, the decellularized extracellular matrix is ​​treated with a high-speed homogenizer at 8000-20000 rpm for 1-10 min in a moist state, and then graded through a sieve with a pore size of 50-200 μm to obtain ovarian decellularized extracellular matrix microfilaments with a length of 50-150 μm and a diameter of 1-30 μm.

[0039] Furthermore, in step B5, the length of the ovarian decellularized extracellular matrix microfilaments is controlled by the sieve grading aperture, and the diameter is controlled by the processing speed and time parameters of the high-speed homogenizer or ball mill.

[0040] Furthermore, the shearing or grinding is carried out under frozen or humid conditions, using a high-speed homogenizer at 8000-20000 rpm for 1-10 min, or a ball mill at 200-600 rpm for 5-60 min, and then graded by sieve to obtain the length range. Furthermore, the mesenchymal stem cells derived from menstrual blood simultaneously meet the following phenotypic criteria: CD73 positivity rate not less than 90%, CD90 positivity rate not less than 90%, CD105 positivity rate not more than 90%, CD34 positivity rate not more than 5%, and CD45 positivity rate not more than 5%, wherein the positivity rates are detected by flow cytometry. Furthermore, the hypoxia pretreatment conditions are an oxygen volume fraction of 0.5-5.0 vol%, a treatment time of 12-72 h, and the hypoxia pretreatment is carried out in an incubator at 37℃ and 5 vol% carbon dioxide. After hypoxia pretreatment, the viability of the blood-derived mesenchymal stem cells is not less than 85%.

[0041] Furthermore, the microfluidic device is a flow-focusing or co-flow-focusing microfluidic chip, wherein the narrowest channel width of the microfluidic chip is 200-1000 μm, the dispersed phase flow rate is 0.1-5 mL / h, and the continuous phase flow rate is 1-50 mL / h. Furthermore, in step S3, the final concentration of blood-derived mesenchymal stem cells in the dispersed aqueous phase is 1× -2× The final concentration of ovarian decellularized extracellular matrix microfilaments in the aqueous dispersion phase was 0.1-30 mg / mL.

[0042] Furthermore, the crosslinking reaction in step S3 is carried out at room temperature to 37°C.

[0043] Furthermore, in step S3, the continuous phase is mineral oil.

[0044] Furthermore, in step S3, the micro-organ units are recovered by centrifugation or filtration, and the washing is performed using phosphate buffer solution, with the number of washing cycles being 3-10.

[0045] Furthermore, the injectable microtissue unit formulation is stored at 2-8°C after preparation, with a shelf life of 1-7 days.

[0046] Furthermore, the preparation process is carried out under sterile conditions; the first precursor solution and the second precursor solution are filtered and sterilized through sterile filters with a pore size of 0.22 μm before the addition of blood-derived mesenchymal stem cells; the microfluidic device and the container in contact with the material are pre-sterilized; the final injectable microtissue unit formulation is not subjected to terminal filtration sterilization and is not sterilized by ultraviolet irradiation.

[0047] Furthermore, the long-tube puncture and injection assembly includes a puncture needle or catheter with an inner diameter of 1.0-2.0 mm and a length of not less than 150 mm.

[0048] Beneficial technical effects 1. This invention employs a ternary gel matrix system composed of sodium hyaluronate, four-arm polyethylene glycol maleimide, and thiolated carboxymethyl dextran in the second precursor solution. Sodium hyaluronate imparts excellent fluidity and low viscosity to the matrix before injection, while the maleimide-thio-Michael addition crosslinking network rapidly solidifies in situ after injection. The two are decoupled in time, overcoming the inherent contradiction of traditional single-component hydrogels in achieving both injectability and molding stability. This significantly reduces the resistance of needle injection while ensuring the spatial retention stability of micro-tissue units in the ovarian region.

[0049] 2. This invention prepares injectable micro-tissue unit formulations using a microfluidic device. By utilizing the principles of flow focusing or co-flow focusing, droplet generation is precisely controlled, maintaining the equivalent particle size of the micro-tissue units at 150-800 μm with a particle size variation coefficient not exceeding 15%. This is superior to the particle size distribution level of traditional batch emulsification methods, significantly improving batch-to-batch consistency and laying the foundation for precise control of clinical dosage and reproducibility of therapeutic effects.

[0050] 3. This invention introduces ovarian decellularized extracellular matrix microfilaments with a length of 50-150 μm and a diameter of 1-30 μm as biomimetic fiber reinforcement units of the gel matrix. The ovarian-specific collagen network, glycosaminoglycans and endogenous growth factors retained in them not only provide mechanical support for the fibrous skeleton of the micro-tissue unit and improve shear toughness, but also directly activate the ovarian homing paracrine program of mesenchymal stem cells derived from menstrual blood, significantly improving the integration interface between cells and ovarian tissue, and showing obvious biological signal advantages compared with non-biomimetic gel matrix systems.

[0051] 4. This invention pre-treats menstrual blood-derived mesenchymal stem cells with hypoxia at an oxygen volume fraction of 0.5-5.0 vol% for 12-72 h before encapsulation. This activates the hypoxia-inducible factor signaling pathway in cells and pre-regulates the secretion reserves of repair-promoting paracrine factors such as vascular endothelial growth factor and hepatocyte growth factor. This allows menstrual blood-derived mesenchymal stem cells to exert maximum efficacy in the shortest possible time after injection into the ovary, effectively compensating for the lag in cell function expression during the period from in vivo injection to blood supply reconstruction.

[0052] 5. In this invention, the first precursor solution and the second precursor solution are prepared separately and stored separately after independent sterilization filtration. The two solutions do not come into contact before step S3, which fundamentally eliminates the risk of process runaway caused by premature initiation of the cross-linking reaction. At the same time, the final injectable microtissue unit formulation does not undergo terminal filtration sterilization and ultraviolet irradiation sterilization. The aseptic process throughout the process protects the structural integrity of the microtissue unit and the vitality of blood-derived mesenchymal stem cells, which has significant advantages over traditional cell preparation processes that require terminal sterilization. Attached Figure Description

[0053] Figure 1 The Fourier transform infrared spectra of Example 1 and Comparative Example 7 are shown.

[0054] Figure 2 The images show the oscillatory rheological time scans of Example 1 and Comparative Example 3.

[0055] Figure 3 The images show the Raman spectra of Example 1 and Comparative Example 4.

[0056] Figure 4 The X-ray diffraction patterns are those of Example 1 and Comparative Example 4.

[0057] Figure 5 The following is a time-series secretion curve of vascular endothelial growth factor (VEGF) for Example 1 and Comparative Example 6.

[0058] Figure 6 This is a low-magnification scanning electron microscope image of the micro-organisms prepared in Example 1 of the present invention.

[0059] Figure 7 This is a magnified TEM image of the decellularized extracellular matrix microfilament region of the ovary in the micro-tissue unit of Example 1. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0061] Example 1 This embodiment provides a blood-derived mesenchymal stem cell ovarian targeting complex. The complex includes an injectable microtissue unit formulation composed of multiple microtissue units. The arithmetic mean of the equivalent particle size of the microtissue units is 475 μm, and the coefficient of variation of the equivalent particle size is 12%. The equivalent particle size is the equivalent diameter of the microtissue unit calculated based on the projected area in a microscopic image. The injectable microtissue unit formulation includes blood-derived mesenchymal stem cells, ovarian decellularized extracellular matrix microfilaments, and a gel matrix. The gel matrix contains sodium hyaluronate, four-arm polyethylene glycol maleimide, and thiolated carboxymethyl dextran in a second precursor solution. The four-arm polyethylene glycol maleimide and the thiolated carboxymethyl dextran in the second precursor solution form a thioether cross-linked structure through a maleimide-thiol Michael addition reaction.

[0062] The second precursor solution contains thiolated carboxymethyl dextran, prepared via the following steps: A1. Sodium carboxymethyl dextran with a weight-average molecular weight of 150,000 Da and a degree of substitution of 0.9 is dissolved in 0.10 mol / L MES buffer and the pH is adjusted to 5.5 to obtain a 1.5% (w / v) carboxymethyl dextran sodium salt solution. Under continuous stirring, β-mercaptoethylamine, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide are added to the solution, wherein the mass ratio of sodium carboxymethyl dextran to β-mercaptoethylamine is 1:0.12, and the molar ratio of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1.4:1; A2. At a concentration of 0.10... The reaction was carried out in mol / L MES buffer at pH 5.2 with continuous stirring at 30°C for 12 h; A3, when the thiol content of the obtained polymer on a dry basis was measured to be 0.35 mmol / g by the Ellman reagent method, stirring was stopped and the dialysis post-treatment in step A4 was immediately performed to terminate the coupling reaction; A4, using a dialysis bag with a molecular weight cutoff of 8000 Da, the dialysis was carried out in phosphate buffer at pH 7.0 for 48 h, with the dialysis solution being changed every 10 h. After dialysis, the solution was frozen at -60°C and then freeze-dried at a vacuum of 20 Pa and a temperature of -20°C for 48 h to obtain the thiolized carboxymethyl dextran in the second precursor solution.

[0063] The ovarian decellularized extracellular matrix microfilaments were prepared through the following steps: B1. Porcine ovarian tissue was cut into pieces and washed three times with a 0.9% (w / v) sodium chloride aqueous solution, with each wash volume being 10 times the tissue mass, at a washing temperature of 4°C; B2. The porcine ovarian tissue was placed in a 0.5% (w / v) sodium dodecyl sulfate aqueous solution, with a liquid volume to tissue mass ratio of 20 mL / g, and treated at 25°C on a shaker at 100 rpm for 24 h. Subsequently, it was placed in a 1.0 vol% Triton X-100 aqueous solution, with a liquid volume to tissue mass ratio of 20 mL / g, and treated at 25°C on a shaker at 100 rpm for 12 h. Finally, it was treated with a 0.372% (w / v) sodium chloride solution corresponding to a molar concentration of 10... A1. Chelating and washing with an aqueous solution of ethylenediaminetetraacetic acid disodium salt dihydrate at mmol / L, pH 7.5, 3 times, 30 min each time; B2. The tissue treated in step B2 was placed in an aqueous solution containing deoxyribonuclease I and anhydrous magnesium chloride. The concentration of deoxyribonuclease I was 100 U / mL, and the concentration of anhydrous magnesium chloride was 5 mmol / L, corresponding to a mass-volume percentage concentration of 0.048% (w / v). The pH of the aqueous solution was 7.8, the treatment temperature was 30℃, the treatment time was 3 h, and the liquid volume to tissue mass ratio was 25 mL / g. The anhydrous magnesium chloride was used to provide magnesium ions; B4. The resulting decellularized extracellular matrix met the requirement of a double-stranded deoxyribonucleic acid content of 35%. The content of the double-stranded deoxyribonucleic acid (DRNA) fragments was determined by quantification of nucleic acid (QSIQ) using a nucleic acid quantification method, and the length of the DRNA fragments was determined by gel electrophoresis. B5. The decellularized extracellular matrix was treated with a high-speed homogenizer at 14000 rpm for 5 min under moist conditions, and then graded through a 120 μm sieve to obtain ovarian decellularized extracellular matrix microfilaments with a length of 90 μm and a diameter of 12 μm. The length of the ovarian decellularized extracellular matrix microfilaments was controlled by the sieve grading pore size, and the diameter was controlled by the processing speed and time parameters of the high-speed homogenizer.

[0064] The menstrual blood-derived mesenchymal stem cells are derived from human menstrual blood and meet the following phenotypic criteria: CD73 positivity rate of 95%, CD90 positivity rate of 96%, CD105 positivity rate of 94%, CD34 positivity rate of 2%, and CD45 positivity rate of 2%, with the positivity rates detected by flow cytometry. Before encapsulation, the menstrual blood-derived mesenchymal stem cells underwent hypoxia pretreatment. The hypoxia pretreatment conditions were 2.0 vol% oxygen for 48 h in an incubator at 37°C and 5 vol% carbon dioxide. After hypoxia pretreatment, the viability of the menstrual blood-derived mesenchymal stem cells was 91%.

[0065] The injectable micro-tissue unit formulation is prepared using a microfluidic device, which is a flow-focusing microfluidic chip. The narrowest channel width of the microfluidic chip is 600 μm, the dispersed phase flow rate is 2.0 mL / h, the continuous phase flow rate is 20 mL / h, the arithmetic mean of the equivalent particle size of the micro-tissue unit is 475 μm, and the coefficient of variation of the equivalent particle size is 12% among 200 micro-tissue units randomly selected from the same batch of formulation. The equivalent particle size is the area equivalent diameter d corresponding to the projected area A of the micro-tissue unit in the microscopic image, where d = (4A / π)^0.5.

[0066] The first precursor solution contains 1.2% (w / v) sodium hyaluronate and 4.0% (w / v) tetra-arm polyethylene glycol maleimide. The second precursor solution contains 1.7% (w / v) thiolated carboxymethyl dextran. The tetra-arm polyethylene glycol maleimide has a molecular weight of 25,000 Da and a terminal maleimide group substitution rate of 93%. The content of the ovarian decellularized extracellular matrix microfilaments in the injectable microtissue unit formulation is 12 mg / mL.

[0067] The gel matrix contains maleimide groups provided by the four-arm polyethylene glycol maleimide and thiol groups provided by the thiolized carboxymethyl dextran in the second precursor solution, with a molar ratio of maleimide groups to thiol groups of 1.0:1. The molar number of maleimide groups is calculated based on the feed mass, molecular weight, four-arm functionality, and substitution rate of the terminal maleimide groups of the four-arm polyethylene glycol maleimide. The molar number of thiol groups is calculated based on the feed mass of the thiolized carboxymethyl dextran in the second precursor solution and the thiol content on a dry basis. The sodium hyaluronate exists in a physically embedded manner within the thioether crosslinking network formed by the four-arm polyethylene glycol maleimide and the thiolized carboxymethyl dextran in the second precursor solution.

[0068] The preparation method of the ovarian-targeting complex derived from menstrual blood mesenchymal stem cells in this embodiment includes the following steps: S1, providing thiolated carboxymethyl dextran and ovarian decellularized extracellular matrix microfilaments in the second precursor solution; S2, preparing the gel matrix precursor: preparing a first precursor solution containing sodium hyaluronate and tetra-arm polyethylene glycol maleimide and a second precursor solution containing thiolated carboxymethyl dextran in the second precursor solution, wherein the first precursor solution and the second precursor solution are stored separately before step S3; wherein the mass-volume percentage concentration of sodium hyaluronate in the first precursor solution is 1.2%. The first precursor solution contains 4.0% (w / v) of tetra-armed polyethylene glycol maleimide (PEG-maleimide) by mass volume; the second precursor solution contains 1.7% (w / v) of thiolated carboxymethyl dextran by mass volume. The first precursor solution is prepared by dissolving sodium hyaluronate and tetra-armed PEG-maleimide in phosphate buffer at pH 7.0. The second precursor solution is prepared by dissolving the thiolated carboxymethyl dextran in phosphate buffer at pH 7.0. The first precursor solution is sealed and stored at 4°C after preparation and is disinfected within 10 days after preparation. The second precursor solution is used in step S3 within 20 h after preparation; after preparation, the second precursor solution is sealed and stored at 4℃ and used in step S3 within 20 h after preparation; S3, Composite and Unitization: Under aseptic conditions, mesenchymal stem cells from menstrual blood and decellularized extracellular matrix microfilaments from the ovary are added to the second precursor solution and mixed to obtain a dispersed aqueous phase. The first precursor solution and the dispersed aqueous phase containing mesenchymal stem cells from menstrual blood and decellularized extracellular matrix microfilaments are contacted and mixed through a microfluidic device to form micro-tissue units. The four-armed polyethylene glycol maleimide undergoes a maleimide-mercapto-Michael addition reaction with the thiolated carboxymethyl dextran in the second precursor solution. The crosslinking time is 8 min from the time the first precursor solution and the dispersed aqueous phase are contacted and mixed in the microfluidic device. The crosslinking reaction is carried out at 25℃, and the continuous phase is mineral oil. After crosslinking, the obtained micro-tissue units are recovered from the continuous phase by centrifugation and washed 5 times with phosphate buffer until no visible oil droplets are visible in the supernatant after washing and the mixture is allowed to stand for 10 h. No oil phase separation occurs, yielding an injectable micro-tissue unit formulation. In step S3, the final concentration of blood-derived mesenchymal stem cells in the dispersed aqueous phase is 8 × 10⁻⁶. The final concentration of ovarian decellularized extracellular matrix microfilaments in the aqueous dispersion phase was 12 mg / mL.

[0069] The preparation process is carried out under aseptic conditions. The first and second precursor solutions are filtered through sterile filters with a pore size of 0.22 μm before the addition of blood-derived mesenchymal stem cells. The microfluidic device and the container in contact with the materials are pre-sterilized. The final injectable microtissue unit formulation is not subjected to terminal filtration sterilization or ultraviolet irradiation sterilization. The injectable microtissue unit formulation is stored at 4°C after preparation for 5 days.

[0070] This embodiment also provides a kit comprising: a) the above-mentioned injectable microtissue unit formulation; b) a long-tube puncture injection assembly, wherein the long-tube puncture injection assembly includes a puncture needle with an inner diameter of 1.5 mm and a length of 180 mm.

[0071] Example 2 This embodiment provides a blood-derived mesenchymal stem cell ovarian-targeting complex. The complex includes an injectable micro-tissue unit formulation, which is composed of multiple micro-tissue units. The arithmetic mean of the equivalent particle size of the micro-tissue units is 350 μm, and the coefficient of variation of the equivalent particle size is 10%. The equivalent particle size is the equivalent diameter of the micro-tissue unit calculated based on the projected area in a microscopic image. The injectable micro-tissue unit formulation includes blood-derived mesenchymal stem cells, ovarian decellularized extracellular matrix microfilaments, and a gel matrix. The gel matrix contains sodium hyaluronate, four-arm polyethylene glycol maleimide, and thiolated carboxymethyl dextran in a second precursor solution. The four-arm polyethylene glycol maleimide and the thiolated carboxymethyl dextran in the second precursor solution form a thioether cross-linked structure through a maleimide-thiol Michael addition reaction.

[0072] The second precursor solution contains thiolated carboxymethyl dextran, prepared via the following steps: A1. Sodium carboxymethyl dextran with a weight-average molecular weight of 350,000 Da and a degree of substitution of 1.0 is dissolved in a 0.15 mol / L phosphate buffer solution and the pH is adjusted to 5.8 to obtain a 2.2% (w / v) carboxymethyl dextran sodium salt solution. Under continuous stirring, β-mercaptoethylamine, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide are added to the solution, wherein the mass ratio of sodium carboxymethyl dextran to β-mercaptoethylamine is 1:0.22, and the molar ratio of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1.7:1; A2. At a concentration of 0.15... The reaction was carried out at 35°C for 18 h in mol / L phosphate buffer at pH 5.5 with continuous stirring; A3, when the thiol content of the obtained polymer on a dry basis was measured by iodometric titration to be 0.45 mmol / g, stirring was stopped and the post-dialysis treatment in step A4 was immediately initiated to terminate the coupling reaction; A4, using a dialysis bag with a molecular weight cutoff of 10000 Da, the mixture was dialyzed in phosphate buffer at pH 7.2 for 60 h, with the dialysate changed every 12 h. After dialyzing, the mixture was frozen at -70°C and then freeze-dried at a vacuum of 15 Pa and a temperature of -20°C for 60 h to obtain the thiolized carboxymethyl dextran in the second precursor solution.

[0073] The ovarian decellularized extracellular matrix microfilaments were prepared through the following steps: B1. Porcine ovarian tissue was cut into pieces and washed four times with a 0.9% (w / v) sodium chloride aqueous solution, with each wash volume being 15 times the tissue mass, at a washing temperature of 10°C; B2. The porcine ovarian tissue was placed in a 0.8% (w / v) sodium dodecyl sulfate aqueous solution, with a liquid volume to tissue mass ratio of 35 mL / g, and treated at 120 rpm on a shaker at 30°C for 18 h. Subsequently, it was placed in a 1.5 vol% Triton X-100 aqueous solution, with a liquid volume to tissue mass ratio of 35 mL / g, and treated at 120 rpm on a shaker at 30°C for 8 h. Finally, it was treated with a 0.558% (w / v) sodium chloride solution corresponding to a molar concentration of 15... Chelating and washing were performed using an aqueous solution of ethylenediaminetetraacetic acid disodium salt dihydrate at a concentration of mmol / L, pH 7.6, for 4 washes, each wash lasting 40 min; B3, the tissue treated in step B2 was placed in an aqueous solution containing deoxyribonuclease I and anhydrous magnesium chloride. The concentration of deoxyribonuclease I was 160 U / mL, and the concentration of anhydrous magnesium chloride was 8 mmol / L, corresponding to a volume percentage concentration of 0.076% (w / v). The pH of the aqueous solution was 7.9, the treatment temperature was 35℃, the treatment time was 2.5 h, and the liquid volume to tissue mass ratio was 35 mL / g. The anhydrous magnesium chloride was used to provide magnesium ions; B4, the resulting decellularized extracellular matrix had a double-stranded deoxyribonucleic acid content of 28 The content of the double-stranded deoxyribonucleic acid (DREE) was determined by nucleic acid quantification, and the length of the DREE fragment was determined by equivalent fragment length analysis. B5. The decellularized extracellular matrix was treated with a high-speed homogenizer at 18000 rpm for 3 min under moist conditions, and then graded through an 80 μm sieve to obtain ovarian decellularized extracellular matrix microfilaments with a length of 65 μm and a diameter of 8 μm. The length of the ovarian decellularized extracellular matrix microfilaments was controlled by the sieve grading pore size, and the diameter was controlled by the processing speed and time parameters of the high-speed homogenizer.

[0074] The menstrual blood-derived mesenchymal stem cells are derived from human menstrual blood and meet the following phenotypic criteria: CD73 positivity rate of 97%, CD90 positivity rate of 98%, CD105 positivity rate of 96%, CD34 positivity rate of 1%, and CD45 positivity rate of 1%. The positivity rates were detected by flow cytometry. Before encapsulation, the menstrual blood-derived mesenchymal stem cells underwent hypoxia pretreatment. The hypoxia pretreatment conditions were 1.0 vol% oxygen and 60 h. The hypoxia pretreatment was carried out in an incubator at 37°C and 5 vol% carbon dioxide. After hypoxia pretreatment, the viability of the menstrual blood-derived mesenchymal stem cells was 93%.

[0075] The injectable micro-tissue unit formulation is prepared using a microfluidic device, which is a co-current focusing microfluidic chip. The narrowest channel width of the microfluidic chip is 350 μm, the dispersed phase flow rate is 0.8 mL / h, the continuous phase flow rate is 35 mL / h, the arithmetic mean of the equivalent particle size of the micro-tissue unit is 350 μm, and the coefficient of variation of the equivalent particle size is 10% among 220 micro-tissue units randomly selected from the same batch of formulation. The equivalent particle size is the area equivalent diameter d corresponding to the projected area A of the micro-tissue unit in the microscopic image, where d = (4A / π)^0.5.

[0076] The first precursor solution contains 0.8% (w / v) sodium hyaluronate and 6.0% (w / v) tetra-arm polyethylene glycol maleimide. The second precursor solution contains 1.8% (w / v) thiolated carboxymethyl dextran. The tetra-arm polyethylene glycol maleimide has a molecular weight of 30,000 Da and a terminal maleimide group substitution rate of 95%. The content of the ovarian decellularized extracellular matrix microfilaments in the injectable microtissue unit formulation is 22 mg / mL.

[0077] The gel matrix contains maleimide groups (based on the number of maleimide groups provided by the four-arm polyethylene glycol maleimide) and thiol groups (based on the number of thiol groups provided by the thiolized carboxymethyl dextran in the second precursor solution), with a molar ratio of maleimide groups to thiol groups of 0.94:1. The molar number of maleimide groups is calculated based on the feed mass, molecular weight, four-arm functionality, and substitution rate of the terminal maleimide groups of the four-arm polyethylene glycol maleimide. The molar number of thiol groups is calculated based on the feed mass of the thiolized carboxymethyl dextran in the second precursor solution and the thiol content on a dry basis. Sodium hyaluronate exists in a physically embedded manner within the thioether crosslinking network formed by the four-arm polyethylene glycol maleimide and the thiolized carboxymethyl dextran in the second precursor solution.

[0078] The preparation method of the ovarian-targeted complex derived from menstrual blood mesenchymal stem cells in this embodiment includes the following steps: S1, providing thiolated carboxymethyl dextran and ovarian decellularized extracellular matrix microfilaments in the second precursor solution; S2, preparing the gel matrix precursor: preparing a first precursor solution containing sodium hyaluronate and tetra-arm polyethylene glycol maleimide and a second precursor solution containing thiolated carboxymethyl dextran in the second precursor solution, wherein the first precursor solution and the second precursor solution are stored separately before step S3; wherein the mass-volume percentage concentration of sodium hyaluronate in the first precursor solution is 0.8%. The first precursor solution contains 6.0% (w / v) of tetra-armed polyethylene glycol maleimide (PEG-maleimide) by mass volume; the second precursor solution contains 1.8% (w / v) of thiolated carboxymethyl dextran by mass volume. The first precursor solution is prepared by dissolving sodium hyaluronate and tetra-armed PEG-maleimide in phosphate buffer at pH 7.2. The second precursor solution is prepared by dissolving the thiolated carboxymethyl dextran in phosphate buffer at pH 7.2. The first precursor solution is sealed and stored at 4°C after preparation, and the solution is tested 8 days after preparation. The second precursor solution is used in step S3 within 18 hours after preparation; after preparation, the second precursor solution is sealed and stored at 4°C and used in step S3 within 18 hours after preparation; S3, Composite and Unitization: Under aseptic conditions, mesenchymal stem cells from menstrual blood and decellularized extracellular matrix microfilaments from the ovary are added to the second precursor solution and mixed to obtain a dispersed aqueous phase. The first precursor solution and the dispersed aqueous phase containing mesenchymal stem cells from menstrual blood and decellularized extracellular matrix microfilaments are contacted and mixed through a microfluidic device to form micro-tissue units. The four-armed polyethylene glycol maleimide undergoes a maleimide-mercapto-Michael addition reaction with the thiolated carboxymethyl dextran in the second precursor solution. The crosslinking time is 5 min from the time the first precursor solution and the dispersed aqueous phase are contacted and mixed in the microfluidic device. The crosslinking reaction is carried out at 30°C. The continuous phase is mineral oil. After crosslinking, the obtained micro-tissue units are recovered from the continuous phase by filtration and washed 6 times with phosphate buffer until no visible oil droplets are found in the supernatant after washing and the mixture is allowed to stand for 10 minutes. No oil phase separation occurred, yielding an injectable micro-tissue unit formulation. In step S3, the final concentration of blood-derived mesenchymal stem cells in the dispersed aqueous phase was 1.5 × 10⁻⁶. The final concentration of ovarian decellularized extracellular matrix microfilaments in the aqueous dispersion phase was 22 mg / mL.

[0079] The preparation process is carried out under aseptic conditions. The first and second precursor solutions are filtered through sterile filters with a pore size of 0.22 μm before the addition of blood-derived mesenchymal stem cells. The microfluidic device and the container in contact with the materials are pre-sterilized. The final injectable microtissue unit formulation is not subjected to terminal filtration sterilization or ultraviolet irradiation sterilization. The injectable microtissue unit formulation is stored at 6°C after preparation for 3 days.

[0080] This embodiment also provides a kit comprising: a) the above-mentioned injectable microtissue unit preparation; b) a long-tube puncture injection assembly, wherein the long-tube puncture injection assembly includes a puncture catheter with an inner diameter of 1.2 mm and a length of 200 mm.

[0081] Example 3 This embodiment provides a blood-derived mesenchymal stem cell ovarian targeting complex. The complex includes an injectable microtissue unit formulation composed of multiple microtissue units. The arithmetic mean of the equivalent particle size of the microtissue units is 600 μm, and the coefficient of variation of the equivalent particle size is 13%. The equivalent particle size is the equivalent diameter of the microtissue unit calculated based on the projected area in a microscopic image. The injectable microtissue unit formulation includes blood-derived mesenchymal stem cells, ovarian decellularized extracellular matrix microfilaments, and a gel matrix. The gel matrix contains sodium hyaluronate, four-arm polyethylene glycol maleimide, and thiolated carboxymethyl dextran in a second precursor solution. The four-arm polyethylene glycol maleimide and the thiolated carboxymethyl dextran in the second precursor solution form a thioether cross-linked structure through a maleimide-thiol Michael addition reaction.

[0082] The second precursor solution contains thiolated carboxymethyl dextran, prepared via the following steps: A1. Sodium carboxymethyl dextran with a weight-average molecular weight of 80,000 Da and a degree of substitution of 0.75 is dissolved in 0.08 mol / L MES buffer and the pH is adjusted to 5.0 to obtain a 1.0% (w / v) carboxymethyl dextran sodium salt solution. Under continuous stirring, β-mercaptoethylamine, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide are added to the solution, wherein the mass ratio of sodium carboxymethyl dextran to β-mercaptoethylamine is 1:0.08, and the molar ratio of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1.2:1; A2. At a concentration of 0.08... The reaction was carried out in mol / L MES buffer at pH 4.8 with continuous stirring at 25°C for 8 h; A3, when the thiol content of the obtained polymer on a dry basis was measured to be 0.18 mmol / g by the Ellman reagent method, stirring was stopped and the dialysis post-treatment in step A4 was immediately performed to terminate the coupling reaction; A4, using a dialysis bag with a molecular weight cutoff of 5000 Da, the polymer was dialyzed in phosphate buffer at pH 6.5 for 36 h, with the dialysate being replaced every 8 h. After dialysis, the polymer was vacuum dried at 70 Pa and 50°C for 24 h to obtain the thiolized carboxymethyl dextran in the second precursor solution.

[0083] The ovarian decellularized extracellular matrix microfilaments were prepared through the following steps: B1. Porcine ovarian tissue was cut into pieces and washed twice with a 0.9% (w / v) sodium chloride aqueous solution, with each wash volume being 8 times the tissue mass, at a washing temperature of 15°C; B2. The porcine ovarian tissue was placed in a 0.3% (w / v) sodium dodecyl sulfate aqueous solution, with a liquid volume to tissue mass ratio of 15 mL / g, and treated at 28°C on a shaker at 80 rpm for 32 h. Subsequently, it was placed in a 0.8 vol% Triton X-100 aqueous solution, with a liquid volume to tissue mass ratio of 15 mL / g, and treated at 28°C on a shaker at 80 rpm for 15 h. Finally, it was treated with a 0.186% (w / v) sodium chloride solution corresponding to a molar concentration of 5... Chelating and washing were performed using an aqueous solution of ethylenediaminetetraacetic acid disodium salt dihydrate at a concentration of mmol / L, pH 7.2, three times, with each wash lasting 25 min; B3, the tissue treated in step B2 was placed in an aqueous solution containing deoxyribonuclease I and anhydrous magnesium chloride. The concentration of deoxyribonuclease I was 60 U / mL, and the concentration of anhydrous magnesium chloride was 3 mmol / L, corresponding to a volume percentage concentration of 0.029% (w / v). The pH of the aqueous solution was 7.7, the treatment temperature was 28℃, the treatment time was 4.5 h, and the liquid volume to tissue mass ratio was 18 mL / g. The anhydrous magnesium chloride was used to provide magnesium ions; B4, the resulting decellularized extracellular matrix had a double-stranded deoxyribonucleic acid content of 42%. The content of the double-stranded deoxyribonucleic acid (DREE) was determined by nucleic acid quantification, and the length of the DREE fragment was determined by gel electrophoresis. B5. The decellularized extracellular matrix was treated with a high-speed homogenizer at 10,000 rpm for 7 min under moist conditions, and then graded through a 140 μm sieve to obtain ovarian decellularized extracellular matrix microfilaments with a length of 110 μm and a diameter of 18 μm. The length of the ovarian decellularized extracellular matrix microfilaments was controlled by the sieve grading pore size, and the diameter was controlled by the processing speed and time parameters of the high-speed homogenizer.

[0084] The menstrual blood-derived mesenchymal stem cells are derived from human menstrual blood and meet the following phenotypic criteria: CD73 positivity rate of 93%, CD90 positivity rate of 94%, CD105 positivity rate of 92%, CD34 positivity rate of 3%, and CD45 positivity rate of 3%, with the positivity rates detected by flow cytometry. Before encapsulation, the menstrual blood-derived mesenchymal stem cells underwent hypoxia pretreatment. The hypoxia pretreatment conditions were 4.0 vol% oxygen for 24 h in an incubator at 37°C and 5 vol% carbon dioxide. After hypoxia pretreatment, the viability of the menstrual blood-derived mesenchymal stem cells was 88%.

[0085] The injectable micro-tissue unit formulation is prepared using a microfluidic device, which is a flow-focusing microfluidic chip. The narrowest channel width of the microfluidic chip is 850 μm, the dispersed phase flow rate is 3.5 mL / h, the continuous phase flow rate is 12 mL / h, the arithmetic mean of the equivalent particle size of the micro-tissue unit is 600 μm, and the coefficient of variation of the equivalent particle size is 13% among 210 micro-tissue units randomly selected from the same batch of formulation. The equivalent particle size is the area equivalent diameter d corresponding to the projected area A of the micro-tissue unit in the microscopic image, where d = (4A / π)^0.5.

[0086] The first precursor solution contains 1.7% (w / v) sodium hyaluronate and 3.5% (w / v) tetra-arm polyethylene glycol maleimide. The second precursor solution contains 1.5% (w / v) thiolated carboxymethyl dextran. The tetra-arm polyethylene glycol maleimide has a molecular weight of 30,000 Da and a 90% substitution rate of the terminal maleimide groups. The content of the ovarian decellularized extracellular matrix microfilaments in the injectable microtissue unit formulation is 6 mg / mL.

[0087] The gel matrix contains maleimide groups (based on the number of maleimide groups provided by the four-arm polyethylene glycol maleimide) and thiol groups (based on the number of thiol groups provided by the thiolized carboxymethyl dextran in the second precursor solution), with a molar ratio of maleimide groups to thiol groups of 0.93:1. The molar number of maleimide groups is calculated based on the feed mass, molecular weight, four-arm functionality, and substitution rate of the terminal maleimide groups of the four-arm polyethylene glycol maleimide. The molar number of thiol groups is calculated based on the feed mass of the thiolized carboxymethyl dextran in the second precursor solution and the thiol content on a dry basis. Sodium hyaluronate exists in a physically embedded manner within the thioether crosslinking network formed by the four-arm polyethylene glycol maleimide and the thiolized carboxymethyl dextran in the second precursor solution.

[0088] The preparation method of the ovarian-targeted complex derived from menstrual blood mesenchymal stem cells in this embodiment includes the following steps: S1, providing thiolated carboxymethyl dextran and ovarian decellularized extracellular matrix microfilaments in the second precursor solution; S2, preparing the gel matrix precursor: preparing a first precursor solution containing sodium hyaluronate and tetra-armed polyethylene glycol maleimide and a second precursor solution containing thiolated carboxymethyl dextran in the second precursor solution, wherein the first precursor solution and the second precursor solution are stored separately before step S3; wherein the mass-volume percentage concentration of sodium hyaluronate in the first precursor solution is 1%. The first precursor solution contains 3.5% (w / v) of tetra-arm polyethylene glycol maleimide and 1.5% (w / v) of thiolated carboxymethyl dextran. The second precursor solution is prepared by dissolving sodium hyaluronate and tetra-arm polyethylene glycol maleimide in deionized water at pH 6.8. The second precursor solution is prepared by dissolving the thiolated carboxymethyl dextran in deionized water at pH 6.8. The first precursor solution is sealed and stored at 4°C after preparation, and 11 days after preparation... The second precursor solution is used in step S3 within 22 hours after preparation; after preparation, the second precursor solution is sealed and stored at 4°C and used in step S3 within 22 hours after preparation; S3, Composite and Unitization: Under aseptic conditions, mesenchymal stem cells derived from menstrual blood and decellularized extracellular matrix microfilaments of the ovary are added to the second precursor solution and mixed to obtain a dispersed aqueous phase. The first precursor solution and the dispersed aqueous phase containing mesenchymal stem cells derived from menstrual blood and decellularized extracellular matrix microfilaments of the ovary are contacted and mixed through a microfluidic device to form micro-tissue units. The four-armed polyethylene glycol maleimide undergoes a maleimide-mercapto-Michael addition reaction with the thiolated carboxymethyl dextran in the second precursor solution. The crosslinking time is 12 minutes from the time the first precursor solution and the dispersed aqueous phase are contacted and mixed in the microfluidic device. The crosslinking reaction is carried out at 28°C. The continuous phase is mineral oil. After crosslinking, the obtained micro-tissue units are recovered from the continuous phase by centrifugation and washed 4 times with phosphate buffer until no visible oil droplets are found in the supernatant after washing and the mixture is allowed to stand for 10 minutes. No oil phase separation occurs, yielding an injectable micro-tissue unit formulation. In step S3, the final concentration of blood-derived mesenchymal stem cells in the dispersed aqueous phase is 3 × 10⁻⁶. The final concentration of ovarian decellularized extracellular matrix microfilaments in the aqueous dispersion phase was 6 mg / mL.

[0089] The preparation process is carried out under aseptic conditions. The first and second precursor solutions are filtered through sterile filters with a pore size of 0.22 μm before the addition of blood-derived mesenchymal stem cells. The microfluidic device and the container in contact with the materials are pre-sterilized. The final injectable microtissue unit formulation is not subjected to terminal filtration sterilization or ultraviolet irradiation sterilization. The injectable microtissue unit formulation is stored at 7°C after preparation and has a shelf life of 6 days.

[0090] This embodiment also provides a kit comprising: a) the above-mentioned injectable microtissue unit preparation; b) a long-tube puncture injection assembly, wherein the long-tube puncture injection assembly includes a puncture needle with an inner diameter of 1.8 mm and a length of 160 mm.

[0091] Example 4 This embodiment provides a blood-derived mesenchymal stem cell ovarian targeting complex. The complex includes an injectable microtissue unit formulation composed of multiple microtissue units. The arithmetic mean of the equivalent particle size of the microtissue units is 730 μm, and the coefficient of variation of the equivalent particle size is 14%. The equivalent particle size is the equivalent diameter of the microtissue unit calculated based on the projected area in a microscopic image. The injectable microtissue unit formulation includes blood-derived mesenchymal stem cells, ovarian decellularized extracellular matrix microfilaments, and a gel matrix. The gel matrix contains sodium hyaluronate, four-arm polyethylene glycol maleimide, and thiolated carboxymethyl dextran in a second precursor solution. The four-arm polyethylene glycol maleimide and the thiolated carboxymethyl dextran in the second precursor solution form a thioether cross-linked structure through a maleimide-thiol Michael addition reaction.

[0092] The second precursor solution contains thiolated carboxymethyl dextran, prepared via the following steps: A1. Sodium carboxymethyl dextran with a weight-average molecular weight of 450,000 Da and a degree of substitution of 1.15 is dissolved in a 0.18 mol / L phosphate buffer solution and the pH is adjusted to 5.9 to obtain a 2.8% (w / v) carboxymethyl dextran sodium salt solution. Under continuous stirring, β-mercaptoethylamine, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide are added to the solution, wherein the mass ratio of sodium carboxymethyl dextran to β-mercaptoethylamine is 1:0.27, and the molar ratio of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1.9:1; A2. At a concentration of 0.18... The reaction was carried out at 38°C for 22 h in 1 mol / L phosphate buffer solution at pH 5.7 with continuous stirring; A3, when the thiol content of the obtained polymer on a dry basis was measured by iodometric titration to be 0.48 mmol / g, stirring was stopped and the post-dialysis treatment in step A4 was immediately initiated to terminate the coupling reaction; A4, using a dialysis bag with a molecular weight cutoff of 12000 Da, the polymer was dialyzed in phosphate buffer solution at pH 7.5 for 66 h, with the dialysate being replaced every 11 h. After dialysis, the polymer was frozen at -75°C and then freeze-dried at a vacuum of 12 Pa and a temperature of -20°C for 66 h to obtain the thiolized carboxymethyl dextran in the second precursor solution.

[0093] The ovarian decellularized extracellular matrix microfilaments were prepared through the following steps: B1. Porcine ovarian tissue was cut into pieces and washed five times with a 0.9% (w / v) sodium chloride aqueous solution, with each wash volume being 18 times the tissue mass, at a washing temperature of 20°C; B2. The porcine ovarian tissue was placed in a 0.95% (w / v) sodium dodecyl sulfate aqueous solution, with a liquid volume to tissue mass ratio of 45 mL / g, and treated at 35°C on a shaker at 140 rpm for 10 h. Subsequently, it was placed in a 1.85 vol% Triton X-100 aqueous solution, with a liquid volume to tissue mass ratio of 45 mL / g, and treated at 35°C on a shaker at 140 rpm for 5 h. Finally, it was treated with a 0.707% (w / v) sodium chloride solution corresponding to a molar concentration of 19... Chelating and washing were performed using an aqueous solution of ethylenediaminetetraacetic acid disodium salt dihydrate at a concentration of mmol / L, pH 7.8, for 5 washes, each wash lasting 50 min; B3, the tissue treated in step B2 was placed in an aqueous solution containing deoxyribonuclease I and anhydrous magnesium chloride. The concentration of deoxyribonuclease I was 185 U / mL, and the concentration of anhydrous magnesium chloride was 9.5 mmol / L, corresponding to a volume percentage concentration of 0.091% (w / v). The pH of the aqueous solution was 8.0, the treatment temperature was 36℃, the treatment time was 1.5 h, and the liquid volume to tissue mass ratio was 45 mL / g. The anhydrous magnesium chloride was used to provide magnesium ions; B4, the resulting decellularized extracellular matrix had a double-stranded deoxyribonucleic acid content of 22. The content of the double-stranded deoxyribonucleic acid (DRNA) was determined by nucleic acid quantification using a ng / mg dry weight method, and the length of the DRNA fragment was determined by equivalent fragment length analysis. B5. The decellularized extracellular matrix was treated with a high-speed homogenizer at 19000 rpm for 2 min under moist conditions, and then graded through a 70 μm sieve to obtain ovarian decellularized extracellular matrix microfilaments with a length of 55 μm and a diameter of 4 μm. The length of the ovarian decellularized extracellular matrix microfilaments was controlled by the sieve grading pore size, and the diameter was controlled by the processing speed and time parameters of the high-speed homogenizer.

[0094] The menstrual blood-derived mesenchymal stem cells are derived from human menstrual blood and meet the following phenotypic criteria: CD73 positivity rate of 98%, CD90 positivity rate of 99%, CD105 positivity rate of 97%, CD34 positivity rate of 0.5%, and CD45 positivity rate of 0.5%, with the positivity rates detected by flow cytometry. Before encapsulation, the menstrual blood-derived mesenchymal stem cells underwent hypoxia pretreatment at an oxygen volume fraction of 0.8 vol% for 66 h in an incubator at 37°C and 5 vol% carbon dioxide. After hypoxia pretreatment, the viability of the menstrual blood-derived mesenchymal stem cells was 95%.

[0095] The injectable micro-tissue unit formulation is prepared using a microfluidic device, which is a co-current focusing microfluidic chip. The narrowest channel width of the microfluidic chip is 920 μm, the dispersed phase flow rate is 4.5 mL / h, the continuous phase flow rate is 45 mL / h, the arithmetic mean of the equivalent particle size of the micro-tissue unit is 730 μm, and the coefficient of variation of the equivalent particle size is 14% among 250 micro-tissue units randomly selected from the same batch of formulation. The equivalent particle size is the area equivalent diameter d corresponding to the projected area A of the micro-tissue unit in the microscopic image, where d = (4A / π)^0.5.

[0096] The first precursor solution contains 1.85% (w / v) sodium hyaluronate and 6.8% (w / v) tetra-arm polyethylene glycol maleimide. The second precursor solution contains 1.9% (w / v) thiolated carboxymethyl dextran. The tetra-arm polyethylene glycol maleimide has a molecular weight of 36,000 Da and a terminal maleimide group substitution rate of 96%. The content of the ovarian decellularized extracellular matrix microfilaments in the injectable microtissue unit formulation is 27 mg / mL.

[0097] The gel matrix contains maleimide groups (based on the number of maleimide groups provided by the four-arm polyethylene glycol maleimide) and thiol groups (based on the number of thiol groups provided by the thiolized carboxymethyl dextran in the second precursor solution), with a molar ratio of maleimide groups to thiol groups of 0.82:1. The molar number of maleimide groups is calculated based on the feed mass, molecular weight, four-arm functionality, and substitution rate of the terminal maleimide groups of the four-arm polyethylene glycol maleimide. The molar number of thiol groups is calculated based on the feed mass of the thiolized carboxymethyl dextran in the second precursor solution and the thiol content on a dry basis. The sodium hyaluronate exists in a physically embedded manner within the thioether crosslinking network formed by the four-arm polyethylene glycol maleimide and the thiolized carboxymethyl dextran in the second precursor solution.

[0098] The preparation method of the ovarian-targeted complex derived from menstrual blood mesenchymal stem cells in this embodiment includes the following steps: S1, providing thiolated carboxymethyl dextran and ovarian decellularized extracellular matrix microfilaments in the second precursor solution; S2, preparing the gel matrix precursor: preparing a first precursor solution containing sodium hyaluronate and tetra-arm polyethylene glycol maleimide and a second precursor solution containing thiolated carboxymethyl dextran in the second precursor solution, wherein the first precursor solution and the second precursor solution are stored separately before step S3; wherein the mass-volume percentage concentration of sodium hyaluronate in the first precursor solution is 1.8%. The first precursor solution contained 5% (w / v) of tetra-armed polyethylene glycol maleimide at a mass-volume percentage concentration of 6.8% (w / v); the second precursor solution contained 1.9% (w / v) of thiolated carboxymethyl dextran at a mass-volume percentage concentration of 1.9% (w / v). The first precursor solution was prepared by dissolving sodium hyaluronate and tetra-armed polyethylene glycol maleimide in phosphate buffer at pH 7.4. The second precursor solution was prepared by dissolving the thiolated carboxymethyl dextran in phosphate buffer at pH 7.4. The first precursor solution was sealed and stored at 4°C after preparation, and the solution was tested 6 days after preparation. The second precursor solution is used in step S3 within 15 hours after preparation; after preparation, the second precursor solution is sealed and stored at 4°C and used in step S3 within 15 hours after preparation; S3, Composite and Unitization: Under aseptic conditions, mesenchymal stem cells derived from menstrual blood and decellularized extracellular matrix microfilaments of the ovary are added to the second precursor solution and mixed to obtain a dispersed aqueous phase. The first precursor solution and the dispersed aqueous phase containing mesenchymal stem cells derived from menstrual blood and decellularized extracellular matrix microfilaments of the ovary are contacted and mixed through a microfluidic device to form micro-tissue units. The four-armed polyethylene glycol maleimide undergoes a maleimide-mercapto-Michael addition reaction with the thiolated carboxymethyl dextran in the second precursor solution. The crosslinking time is 2.5 min from the time the first precursor solution and the dispersed aqueous phase are contacted and mixed in the microfluidic device. The crosslinking reaction is carried out at 35°C. The continuous phase is mineral oil. After crosslinking, the obtained micro-tissue units are recovered from the continuous phase by filtration and washed 8 times with phosphate buffer until no visible oil droplets are found in the supernatant after washing and the mixture is allowed to stand for 10 minutes. No oil phase separation occurred, yielding an injectable micro-tissue unit formulation. In step S3, the final concentration of blood-derived mesenchymal stem cells in the dispersed aqueous phase was 1.8 × 10⁻⁶. The final concentration of ovarian decellularized extracellular matrix microfilaments in the aqueous dispersion phase was 27 mg / mL.

[0099] The preparation process is carried out under aseptic conditions. The first and second precursor solutions are filtered through sterile filters with a pore size of 0.22 μm before the addition of blood-derived mesenchymal stem cells. The microfluidic device and the container in contact with the materials are pre-sterilized. The final injectable microtissue unit formulation is not subjected to terminal filtration sterilization or ultraviolet irradiation sterilization. The injectable microtissue unit formulation is stored at 3°C ​​after preparation for 2 days.

[0100] This embodiment also provides a kit comprising: a) the above-mentioned injectable microtissue unit preparation; b) a long-tube puncture injection assembly, wherein the long-tube puncture injection assembly includes a puncture catheter with an inner diameter of 1.9 mm and a length of 190 mm.

[0101] Features of Example 1: This example uses moderate parameter configurations: sodium hyaluronate 1.2%, tetra-arm polyethylene glycol maleimide 4.0%, thiolated carboxymethyl dextran 1.7% in the second precursor solution, micro-tissue unit particle size 475 μm, and cell concentration 8× With a gel density of 12 mg / mL and an ECM microfilament content of 12 mg / mL, all parameters are within a suitable range, achieving a good balance between gel mechanical strength, cell activity, and injectability, making it suitable for routine treatment of patients with premature ovarian failure.

[0102] Features of Example 2: This example uses a high cross-linking density formulation. A dense cross-linking network is formed by 6.0% four-arm polyethylene glycol maleimide and 1.8% thiolated carboxymethyl dextran in the second precursor solution. The micro-tissue unit particle size of 350 μm improves injectability, and the cell concentration is 1.5 × 10⁻⁶. The concentration of cells / mL and ECM microfilaments was significantly increased to 22 mg / mL. Hypoxia pretreatment with 1.0% oxygen concentration for 60 hours enhanced stem cell function, strengthened gel mechanical strength, cell loading density and tissue induction ability, and is suitable for intensive treatment of severe ovarian dysfunction.

[0103] Features of Example 3: This example uses a soft, hydrophilic formulation. Sodium hyaluronate (1.7%) and tetra-arm polyethylene glycol maleimide (3.5%) form a loose cross-linked network. The micro-tissue unit particle size of 600 μm increases the unit loading capacity, and the cell concentration is 3× With a moderate reduction in ECM microfilament content to 6 mg / mL and a cross-linking time of 12 min for full gelation, this product emphasizes gel softness, water retention, and biocompatibility. It is suitable for mild treatment of mild to moderate ovarian dysfunction and for promoting the recruitment of endogenous cells.

[0104] Example 4 Features: This example employs a formulation design with multiple parameters close to their limits. Sodium hyaluronate 1.85%, tetra-arm polyethylene glycol maleimide 6.8%, and thiolated carboxymethyl dextran 1.9% from the second precursor solution (molecular weight 36000 Da, thiol content 0.48 mmol / g) form a highly dense cross-linked network. The micro-tissue unit particle size is 730 μm, and the cell concentration is 1.8 × 10⁻⁶. The formula includes 1 / mL of cells / mL, ECM microfilament content of 27 mg / mL, cross-linking time of 2.5 min, and hypoxia pretreatment with 0.8% oxygen concentration for 66 hours, demonstrating the broad applicability of the claims and its suitability for intensive treatment of severe ovarian failure and high-performance research applications.

[0105] Comparative Example 1: Basically the same as Example 1, except that the mass-volume percentage concentration of sodium hyaluronate in the gel matrix is ​​0.3% (w / v), and other conditions remain unchanged.

[0106] Comparative Example 2: Basically the same as Example 1, except that the mass-volume percentage concentration of sodium hyaluronate in the gel matrix is ​​2.5% (w / v), and other conditions remain unchanged.

[0107] Comparative Example 3: Basically the same as Example 1, except that the mass-volume percentage concentration of four-arm polyethylene glycol maleimide in the gel matrix is ​​2.0% (w / v), and other conditions remain unchanged.

[0108] Comparative Example 4: Basically the same as Example 1, except that ovarian decellularized extracellular matrix microfilaments are not added to the second precursor solution in step S3, and other conditions remain unchanged.

[0109] Comparative Example 5: It is basically the same as Example 1, except that in step S3, the crosslinking time of the maleimide-thiol Michael addition reaction is 20 min, starting from the time the first precursor solution and the dispersed aqueous phase come into contact and mix in the microfluidic device, while other conditions remain unchanged.

[0110] Comparative Example 6: It is basically the same as Example 1, except that the mesenchymal stem cells derived from menstrual blood are not pretreated with hypoxia before step S3, but are pre-cultured for 48 h in a normoxic environment at 37°C and 5 vol% carbon dioxide for 20 vol% oxygen volume fraction, while other conditions remain unchanged.

[0111] Comparative Example 7: Basically the same as Example 1, except that the thiolated carboxymethyl dextran in the second precursor solution was replaced with unthiolated sodium carboxymethyl dextran (weight average molecular weight 150,000 Da, degree of substitution 0.9, mass volume percentage concentration 1.7% (w / v), dissolved in phosphate buffer at pH 7.0 to prepare the second precursor solution), with other conditions remaining unchanged.

[0112] Comparative Example 8: Basically the same as Example 1, except that the molecular weight of the four-arm polyethylene glycol maleimide is 5000 Da (the substitution rate of the terminal maleimide group is 93%), and other conditions remain unchanged.

[0113] Performance testing: Experimental Scheme 1: Evaluation of injectability To quantitatively evaluate the steady-state injection resistance of injectable micro-tissue unit formulations through a standard puncture needle with an inner diameter of 1.5 mm and a length of 180 mm, a constant piston speed was used to generate the injection force. The steady-state injection force comprehensively reflects the contribution of the formulation's viscoelasticity and the micro-tissue unit particle size to the resistance of the needle flow. 1 mL of the formulation was placed in a 2 mL standard syringe, a pre-calibrated puncture needle was installed, and the syringe was fixed in a universal testing machine fixture. The piston was pressed down at a constant speed of 2 mm / min, and the mean steady-state injection force was recorded within the displacement range of 20%–80%. Injection force testing was performed according to the relevant procedures: ambient temperature 25±2℃, relative humidity 40%–60%, injection speed 2 mm / min. The mean injection force ± standard deviation was reported in N, with <5 N considered the injectability acceptance threshold.

[0114] Experimental Protocol 2: Immediate Cell Viability Measurement After Injection (Calcechlorin-AM / Propidium Iodide Double Staining Method) To quantify the damage to the viability of encapsulated blood-derived mesenchymal stem cells caused by shear stress during injection, a live / dead cell double staining method was used to detect the effect of the formulation immediately recovered after injection using a standard needle according to the experimental protocol. Live cells hydrolyze calcein-AM (Calcein-AM) using esterases to generate green fluorescence, while the increased permeability of dead cell membranes allows propidium iodide (PI) to intercalate into nucleic acids, emitting red fluorescence. Combined staining of these two methods can quantitatively distinguish between live and dead cells. Immediately after injection and recovery, 200 μL of the suspension was added to 2 μM Calcein-AM and 4 μM PI, and incubated at 37°C in the dark for 15 min. Five random fields of view were collected using an upright fluorescence microscope, with at least 200 cells per field. Excitation / emission were 495 / 517 nm (Calcein-AM) and 535 / 617 nm (PI), respectively. The incubation temperature was 37°C. Cell viability (%) was calculated as: (Number of green fluorescent cells / (Total number of green + red fluorescent cells)) × 100%, and the mean ± standard deviation was reported.

[0115] Experimental Scheme 3: Gelation Kinetics and Determination of Elastic Modulus (Oscillatory Rheology) To quantify the gelation time and final storage modulus G' of the maleimide-thiol Michael addition reaction, and to verify the crosslinking time window and gel mechanical properties, oscillatory rheological measurements were performed on the gel matrix precursor mixture prepared according to the formulation of Example 1 (equal volumes of the first and second precursor solutions were mixed, without cell encapsulation). Under low-strain oscillatory shear mode, the storage modulus G' and dissipation modulus G'' dynamically changed with the gelation process, and the point at which G' and G'' crossed was defined as the gelation point. Immediately after mixing equal volumes of the two precursor solutions, the sample was added to the rheometer cone plate (20 mm diameter, 2° cone angle, 48 μm spacing), sealed with anti-evaporation silicone oil, and continuously scanned for 20 min at a frequency of 1 Hz and a strain of 0.5%, recording G' and G'' every 30 s. The test temperature was 25 ± 0.5 °C, the frequency was 1 Hz, and the strain was 0.5%. The time when G' = G'' and the final G' at 20 min were recorded, and the results are reported as mean ± standard deviation.

[0116] Experimental Scheme 4: Evaluation of the structural stability of in vitro micro-tissue units (image analysis particle size retention method) To evaluate the long-term structural integrity maintenance of micro-organisms in a simulated in vivo liquid environment after injection shearing, dynamic particle size tracking was performed on microsphere suspensions recovered after injection using a standard puncture needle according to the experimental protocol over 72 h. If the cross-linked network mechanical strength is sufficient, the particle size will remain stable; if the cross-linking density is insufficient or swelling / dissolution occurs, the mean equivalent diameter will shift over time. After injection recovery, the microspheres were resuspended in PBS at pH 7.4 and incubated in 48-well plates at 37°C. Samples were taken at 0, 6, 12, 24, 48, and 72 h, and non-overlapping fields were captured using an optical microscope (40× objective). The projected area A of at least 100 micro-organisms at each time point was measured using ImageJ software. The equivalent diameter was calculated using d=(4A / π)^0.5, and its distribution was statistically analyzed. The test procedure was performed using PBS at pH 7.4 and 37°C. The pixel resolution was ≤2 μm / pixel, and the number of batches was ≥3. The particle size retention rate (%) was calculated as the average particle size at time t / the average particle size at time 0 h × 100%. The mean ± standard deviation of each time point was reported.

[0117] Experimental Protocol 5: Assay for Vascular Endothelial Growth Factor Paracrine Function (ELISA) To quantitatively detect the paracrine function of menstrual blood-derived mesenchymal stem cells in a three-dimensional encapsulated environment of micro-tissue units and to evaluate the regulatory effect of hypoxia pretreatment, cells were pretreated at a final concentration of 8× Vascular endothelial growth factor (VEGF) in the in vitro culture supernatant of injectable microtissue unit formulations (units / mL) was detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). The solid-phase capture antibody and the detection antibody respectively recognize different epitopes of human VEGF molecules. HRP-labeled enzyme catalyzes substrate color development, and the absorbance is directly proportional to the VEGF concentration. The microtissue units were then cultured at 8×... The final concentration of [number] cells / mL was placed in a 24-well plate, and 1 mL of serum-free DMEM medium was added. The plate was then incubated at 37°C for 5% [temperature missing]. The culture was carried out, and the supernatant was collected at 24 h, 48 h and 72 h respectively. The absorbance was measured at 450 nm and the background was corrected with 570 nm as the reference wavelength.

[0118] Experimental Scheme Six: Determination of micro-unit particle size and coefficient of variation (static image analysis method) To accurately determine the mean equivalent particle size and coefficient of variation (CV) of injectable microtissue unit formulations from the same batch, verify the microfluidic preparation scheme's ability to control particle size uniformity, and confirm the formulation quality standard of CV ≤ 15%, static image analysis was performed on samples diluted with 1 mL of PBS using 100 μL of the formulation. The equivalent particle size was calculated for the projected area A of each microtissue unit using d = (4A / π)^0.5. After statistical analysis of the population distribution, uniformity was quantified using CV = standard deviation / mean × 100%. Diluted suspensions were spread on a hemocytometer, and non-overlapping fields of view were sequentially acquired using an upright optical microscope (40× objective). The data were imported into ImageJ software, thresholds were set, and the projected area of ​​each microtissue unit was automatically identified and measured. At least 200 units were randomly selected, and their mean, standard deviation, and CV were calculated. Test temperature 25±2℃, pixel resolution ≤2 μm / pixel, count particle number n≥200, repeat 3 batches of samples, report mean particle size ± standard deviation (μm) and CV (%), and take CV≤15% as the qualified standard for formulation.

[0119] Figure 1 The Fourier transform infrared spectra of Example 1 and Comparative Example 7 are shown below, with a test range of wavenumbers from 500 to 3500. The x-axis represents wavenumber, and the y-axis represents absorption intensity. The basic parameter is the full spectrum obtained by scanning at room temperature. The variable parameter is the comparison of the full spectrum obtained by scanning at room temperature in Comparative Example 7, where the thiolized carboxymethyl dextran in the second precursor solution was replaced with unthiolized sodium carboxymethyl dextran, and the rest of the formulation and preparation conditions were the same. The results show that the characteristic peak of the maleimide carbon-carbon double bond in the gel product of Example 1 is about 695. The stretching vibration of the thiol group (SH) is approximately 2550 to 2600. It significantly weakened to the point of disappearing, while at approximately 650 to 700 A new characteristic peak of CS thioether stretching vibration appeared, while Comparative Example 7 did not show the above-mentioned disappearance and new peak characteristics. The conclusion proves that maleimide and thiol undergo specific Michael addition under physiological conditions and form a stable thioether cross-linking network. The characterization path has the rationality of single variable attribution.

[0120] Figure 2The oscillatory rheological time-scan graphs for Example 1 and Comparative Example 3 are shown. The test method was small-strain oscillatory shear time-scan. The basic parameters were: angular frequency 1 Hz, strain 0.5%, test temperature 25℃, scan duration 20 min, and sampling interval 30 s. The horizontal axis represents time, and the vertical axis represents the storage modulus G′ and the dissipation modulus G″. The variable parameter was that in Comparative Example 3, the mass-volume percentage concentration of the four-arm polyethylene glycol maleimide was reduced from 4.0% to 2.0%, while the other conditions remained the same. The results showed that in Example 1, G′ and G″ crossed at about 8 min and quickly entered the high modulus plateau. The final state G′ was about 820 Pa. In Comparative Example 3, the crossing time was significantly delayed, and the final state G′ was only about 245 Pa. The conclusion proves that increasing the concentration of the four-arm polyethylene glycol maleimide can significantly improve the crosslinking density and gelation kinetics and obtain a higher final state elastic modulus, thus supporting the rationality of the controllable crosslinking window of 1 to 15 min and structural stability.

[0121] Figure 3 The Raman spectra of Example 1 and Comparative Example 4 are shown, with the test range being Raman shifts from 800 to 1800. The x-axis represents Raman shift, and the y-axis represents intensity. The basic parameters are the superimposed comparison of spectra obtained under the same test conditions. The variable parameters are Comparative Example 4, which does not add ovarian decellularized extracellular matrix microfilaments; the composition of the rest of the system is consistent with the preparation process. The results show that collagen-related proline characteristic peaks of approximately 855 appeared in Example 1. Amide III band approximately 1245 Amide I band approximately 1665 and between approximately 950 and 1100 The study showed that COC vibrations related to glycosaminoglycans were present, while the peak intensity in Comparative Example 4 was significantly reduced and approached the background. The conclusion is that the extracellular matrix microfilaments of the ovary are preserved in the micro-tissue unit and deliver key components such as collagen and glycosaminoglycans, providing specific biomimetic microenvironment support for cells. The causal relationship of the control selection is clear.

[0122] Figure 4 The X-ray diffraction patterns of Example 1 and Comparative Example 4 are shown. The test range is 2θ angle from 5° to 35°. The horizontal axis is 2θ and the vertical axis is diffraction intensity. The basic parameter is the superposition and comparison of the spectra obtained under the same diffraction conditions. The variable parameter is that Comparative Example 4 does not add ovarian decellularized extracellular matrix microfilaments, and the other conditions are the same. The results show that Example 1 has a low-angle characteristic diffraction signal with collagen fiber axial periodic correlation at about 2θ equals 7.3° and a broad scattering peak near about 2θ equals 20°, while the corresponding characteristics of Comparative Example 4 are significantly weakened or absent. The conclusion proves that the collagen fiber-related ordered structure of ovarian decellularized extracellular matrix microfilaments is preserved during the microfluidic preparation process. The structural layer evidence and the Raman chemical layer evidence are consistent with each other, thereby enhancing the correctness and rationality of the scheme.

[0123] Figure 5 The following are time-series secretion curves of vascular endothelial growth factor (VEGF) for Example 1 and Comparative Example 6. The characterization method was ELISA detection of VEGF content in the culture supernatant. The baseline parameters were sampling at three time points: 24 h, 48 h, and 72 h. Every Cell counts, with time on the x-axis and VEGF secretion on the y-axis. Variables include: Comparative Example 6, which underwent hypoxia pretreatment for 2.0... Change to standard oxygen 20 The remaining gel composition was consistent with the cross-linked structure; the results showed that the VEGF secretion level in Example 1 was higher than that in Comparative Example 6 at all time points, with a difference of approximately 64% at 72 h, corresponding to approximately 385 and 235. Every The difference in VEGF can be attributed to the selective enhancement of paracrine function by hypoxia pretreatment, based on the negative control information showing no significant difference in elastic modulus between the two treatments. The conclusion demonstrates that the functional enhancement is independent of gel mechanical factors, supporting the intrinsic consistency of the design.

[0124] Figure 6 The images shown are low-magnification scanning electron microscope (SEM) images of the microstructure units prepared in Example 1 of this invention. The overall morphology and particle size distribution were characterized using secondary electron mode. The images show that the microstructure exhibits a near-spherical geometry, with an optically statistically average equivalent particle size of 475 μm and a coefficient of variation (CV) of 12%. The particle size is concentrated in the range of 400-600 μm, with only a small number of droplets and abnormally sized particles visible. This characteristic is highly consistent with the expected microfluidic flow focusing to generate droplets and the derived CV value, demonstrating that the microfluidic mechanism can stably prepare near-spherical microstructures with uniform size.

[0125] Figure 7 This is a magnified TEM image of the ovarian decellularized extracellular matrix microfilament region in the micro-tissue unit of Example 1, focusing on the diameter distribution and supramolecular periodic structure of collagen fibrils. At higher magnification, the microfilament region was further resolved into a bundle-like structure composed of numerous collagen fibrils. Statistical analysis showed that the fibril diameters were mainly distributed between 30–100 nm, exhibiting a certain degree of polydispersity. Through detailed observation of local areas, alternating light and dark stripes along the fiber axis could be distinguished, namely the unique D-band periodic structure of collagen fibers, with a measured periodic width of approximately 47–67 nm. This result demonstrates that after decellularization and shearing, the collagen fibrils in the extracellular matrix microfilaments of the ovary still maintain their natural nanoscale diameter distribution and characteristic supramolecular stacking structure. The slight reduction in the periodic value is consistent with the conventional appearance changes after dehydration with anhydrous ethanol and resin embedding.

[0126] Table 1. Performance comparison data between the examples and comparative examples.

[0127] As can be seen from the performance of the examples and comparative examples in Table 1, the performance indicators of Examples 1–4 are comprehensively superior to those of the comparative examples. Insufficient sodium hyaluronate concentration (Comparative Example 1) resulted in a sparse gel skeleton, a significant decrease in elastic modulus, and a simultaneous decline in cell viability and VEGF secretion; excessively high concentration (Comparative Example 2) led to a rapid increase in system viscosity, a significant increase in injection force, and a decrease in cell encapsulation efficiency; insufficient concentration of four-arm polyethylene glycol maleimide (Comparative Example 3) reduced cross-linking density, worsened the particle size variation coefficient, and significantly decreased the gel elastic modulus; removal of ovarian decellularized extracellular matrix microfilaments (Comparative Example 4) had limited impact on injection force and elastic modulus, but significantly reduced cell viability and VEGF secretion after 7 days, revealing that microfilaments are a key biomimetic support element for maintaining stem cell bioactivity; excessively long cross-linking time (Comparative Example 5) led to… Excessive cross-linking led to a sharp increase in injection force and a sudden drop in cell viability. Removing the hypoxia pretreatment (Comparative Example 6) had no significant effect on elastic modulus and particle size uniformity, but paracrine function was selectively weakened, and VEGF secretion decreased by about 39%, demonstrating the specific regulatory effect of hypoxia pretreatment on the maintenance of stem cell function. Replacing it with unthiolized sodium carboxymethyl dextran (Comparative Example 7) prevented the establishment of thioether cross-links, reduced the elastic modulus to the lowest level, and completely destroyed particle size uniformity. The four-arm polyethylene glycol maleimide with too low a molecular weight (Comparative Example 8) had insufficient arm length and a loose network, resulting in a significant decrease in mechanical properties and uniformity, confirming the necessity and synergistic effect of each technical parameter.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A menstrual blood-derived mesenchymal stem cell ovarian-targeting complex, characterized in that, The composite comprises an injectable microtissue unit formulation, which is composed of multiple microtissue units, wherein the arithmetic mean of the equivalent particle size of the microtissue units is 150-800 μm, and the coefficient of variation of the equivalent particle size is not higher than 15%. The equivalent particle size is the equivalent diameter of the micro-tissue unit calculated based on the projected area in a microscopic image; the injectable micro-tissue unit formulation includes blood-derived mesenchymal stem cells, ovarian acellular extracellular matrix microfilaments, and a gel matrix; the gel matrix contains sodium hyaluronate, tetra-arm polyethylene glycol maleimide, and thiolated carboxymethyl dextran in the second precursor solution. The four-armed polyethylene glycol maleimide and the thiolated carboxymethyl dextran in the second precursor solution form a thioether cross-linked structure through a maleimide-thioMichael addition reaction.

2. The composite according to claim 1, characterized in that, The second precursor solution contains thiolated carboxymethyl dextran, which is prepared by the following steps: A1. Mix sodium carboxymethyl dextran, β-mercaptoethylamine, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; A2. Under conditions of pH 4.5-6.0, activate the carboxyl group of sodium carboxymethyl dextran and react it with β-mercaptoethylamine for 2-24 h; A3. The reaction is terminated when the thiol content of the obtained polymer, on a dry basis, is 0.05-0.50 mmol / g; A4. Unreacted small molecules were removed by dialysis, and the thiolated carboxymethyl dextran was obtained after drying.

3. The composite according to claim 1, characterized in that, The decellularized extracellular matrix microfilaments of the ovary were prepared through the following steps: B1. Cut the pig ovarian tissue and wash it with sodium chloride solution; B2. Decellularization treatment: The porcine ovarian tissue was treated in an aqueous solution of sodium dodecyl sulfate with a mass-volume percentage concentration of 0.1-1.0% (w / v), then treated in a Triton X-100 aqueous solution, and then chelated and washed with an aqueous solution containing disodium ethylenediaminetetraacetate dihydrate. B3. Nucleic acid removal: The tissue treated in step B2 is placed in an aqueous solution containing deoxyribonuclease I and anhydrous magnesium chloride, wherein the anhydrous magnesium chloride is used to provide magnesium ions; B4. Endpoint criteria: The obtained decellularized extracellular matrix meets the following requirements: double-stranded deoxyribonucleic acid content not exceeding 50 ng / mg dry weight, deoxyribonucleic acid fragment length not exceeding 200 base pairs, and no visible cell nuclei in the tissue sections; B5. Microfilamentization: The decellularized extracellular matrix is ​​sheared or ground to obtain ovarian decellularized extracellular matrix microfilaments with a length of 50-150 μm and a diameter of 1-30 μm.

4. The composite according to claim 1, characterized in that, The menstrual blood-derived mesenchymal stem cells are derived from human menstrual blood and are CD73 positive, CD90 positive, CD105 positive, CD34 negative, and CD45 negative; the menstrual blood-derived mesenchymal stem cells are pretreated with hypoxia before encapsulation.

5. The composite according to claim 1, characterized in that, The injectable microtissue unit formulation is prepared using a microfluidic device, and the coefficient of variation of the equivalent particle size of the microtissue unit is not higher than 15%.

6. The composite according to claim 1, characterized in that, The first precursor solution contains sodium hyaluronate at a mass-volume percentage concentration of 0.5-2.0% (w / v) and tetra-arm polyethylene glycol maleimide at a mass-volume percentage concentration of 3.0-7.0% (w / v), while the second precursor solution contains thiolated carboxymethyl dextran at a mass-volume percentage concentration of 0.1-2.0% (w / v).

7. The composite according to claim 1, characterized in that, The four-arm polyethylene glycol maleimide has a molecular weight of 10,000-40,000 Da and a substitution rate of not less than 90% for the terminal maleimide groups.

8. The composite according to claim 1, characterized in that, The content of the ovarian decellularized extracellular matrix microfilaments in the injectable microtissue unit formulation is 0.1-30 mg / mL.

9. A method for preparing a menstrual blood-derived mesenchymal stem cell ovarian-targeting complex as described in any one of claims 1-8, characterized in that, The composite is an injectable microtissue unit formulation, comprising the following steps: S1, providing thiolated carboxymethyl dextran and ovarian decellularized extracellular matrix microfilaments in the second precursor solution; S2. Preparation of gel matrix precursor: Prepare a first precursor solution containing sodium hyaluronate and tetra-armed polyethylene glycol maleimide and a second precursor solution containing thiolated carboxymethyl dextran from the second precursor solution. The first precursor solution and the second precursor solution are stored separately before step S3. S3. Combining and Unitization: Menstrual blood-derived mesenchymal stem cells and ovarian decellularized extracellular matrix microfilaments are added to the second precursor solution and mixed. The first precursor solution and the second precursor solution containing menstrual blood-derived mesenchymal stem cells and ovarian decellularized extracellular matrix microfilaments are contacted and mixed through a microfluidic device to form micro-tissue units. The four-armed polyethylene glycol maleimide undergoes a maleimide-mercapto-Michael addition reaction with the thiolated carboxymethyl dextran in the second precursor solution. The crosslinking time is 1-15 min from the time the first precursor solution and the second precursor solution come into contact and mix in the microfluidic device, resulting in an injectable micro-tissue unit formulation.

10. The preparation method according to claim 9, characterized in that, In step S2, the mass-volume percentage concentration of sodium hyaluronate in the first precursor solution is 0.5-2.0% (w / v), and the mass-volume percentage concentration of tetra-arm polyethylene glycol maleimide is 3.0-7.0% (w / v); the mass-volume percentage concentration of thiolated carboxymethyl dextran in the second precursor solution is 0.1-2.0% (w / v).

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