A muscle stem cell resuscitation protection wash, quality assessment kit, and methods of use

Abstract

The application discloses a muscle stem cell resuscitation protection washing liquid, a quality evaluation kit and a use method, and belongs to the technical field of cell biology. In the application, vitamin E succinate and mannose peptide are used as core antioxidant components, and gypenoside, spilanthes acmella merrill polysaccharide, trehalose, L-proline and magnesium sulfate heptahydrate are used to construct a four-in-one resuscitation protection washing liquid with isotonic buffer, extracellular membrane protection, intracellular protein stabilization, antioxidant and mitochondrial stabilization functions; after muscle stem cells are resuscitated, the survival rate of the muscle stem cells is increased to more than 93%. The kit integrates a cell resuscitation protection washing liquid, a cell survival rate, a proliferation rate, a Pax7 / MyoD double-positive stemness evaluation and a cryopreservation damage four-dimension detection system, realizes whole-process closed-loop management of muscle stem cell resuscitation protection and quality evaluation, and makes the double-positive rate of stemness markers more than 90%, thereby providing reliable quality guarantee for cell bank management and clinical application.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology and relates to a muscle stem cell resuscitation and protection washing solution, a quality assessment kit, and a method of use. Background Technology

[0002] Skeletal muscle satellite cells (SMSCs) are adult stem cells that reside between skeletal muscle fibers and the basement membrane. They possess the potential for self-renewal and differentiation into myoblasts and muscle fibers, and have extremely broad application prospects in cell therapy, regenerative medicine, and in vitro muscle model construction for hereditary muscle diseases such as muscle injury repair, Duchenne muscular dystrophy, and spinal muscular atrophy.

[0003] In the clinical-grade preparation and transportation of muscle stem cells, cryopreservation at -196℃ in liquid nitrogen is a crucial and indispensable technique. Cell resuscitation is the process of rapidly thawing cells frozen in liquid nitrogen to restore their normal physiological activity. Its core principle is to reduce damage to cell structure and function and maintain cell viability and stemness by precisely controlling the heating rate, regulating the balance of intracellular and extracellular osmotic pressure, inhibiting oxidative stress and ice crystal damage. During the cryopreservation and resuscitation of muscle stem cells, cells must simultaneously endure multiple forms of damage, including osmotic stress, ice crystal damage, mitochondrial membrane potential instability, and oxidative stress caused by a sudden surge of reactive oxygen species (ROS). This leads to a significant decrease in cell viability and weakened expression of stemness markers (Pax7, MyoD). Therefore, the cryopreservation and resuscitation process directly determines the batch quality of muscle stem cells and restricts the safety of industrial production and clinical application of muscle stem cells.

[0004] Currently, most resuscitation and protective washing solutions for muscle stem cells use PBS (phosphate-buffered saline) or D-Hank solution, which only provide an isotonic environment and lack antioxidant and mitochondrial protection. While some resuscitation solutions add vitamin E, they use common lipid-soluble α-tocopherol, which has extremely poor water solubility and easily precipitates and aggregates in aqueous systems. This results in low effective cell uptake, limited cell membrane penetration, insufficient antioxidant efficiency, and low cell viability. Furthermore, quality assessments, such as quality testing, are necessary after muscle stem cell resuscitation. However, current quality assessments are limited to viability and do not include multi-dimensional comprehensive evaluations of muscle stem cell-specific stem cell markers, proliferative function, and cryopreservation damage. Consequently, it is impossible to accurately determine the functional status of cells after resuscitation, making it difficult to guarantee the safety and effectiveness of subsequent clinical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a muscle stem cell resuscitation and protection washing solution, a quality assessment kit, and a method of use, in order to solve the problem of low cell survival rate when resuscitating muscle stem cells using existing resuscitation and protection washing solutions.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, this application provides a muscle stem cell resuscitation and protective washing solution, which uses sterile deionized water as a solvent and comprises, according to a final concentration: 15-25 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 20-50 μg / mL mannan peptide, 10-30 μg / mL Gynostemma pentaphyllum polysaccharide, 15-40 μg / mL Portulaca oleracea polysaccharide, 120-150 mM sodium chloride, 4.0-7.0 mM potassium chloride, 80-150 mM trehalose, 4-10 mM L-proline, 0.08-0.20 mM vitamin E succinate, and 0.5-1.5 mM magnesium sulfate heptahydrate.

[0007] Secondly, this application provides a muscle stem cell resuscitation quality assessment kit, including the muscle stem cell resuscitation protective washing solution described in the first aspect.

[0008] Thirdly, this application provides a method for using a muscle stem cell resuscitation quality assessment kit, including: The thawed muscle stem cells were slowly added to the muscle stem cell resuscitation and protection washing solution that had been brought to room temperature. After mixing by pipetting, the mixture was centrifuged at 4°C and 1000 r / min for 5 min to obtain cell pellet. The cell pellet was added to fresh muscle stem cell resuscitation and protective washing solution, resuspended by pipetting, and centrifuged at 4°C and 800 r / min for 3 min to obtain resuscitated muscle stem cells. The cell viability, cell proliferation, stem cell expression level, and cryopreservation damage level of the revived muscle stem cells were detected using cell viability assay, cell proliferation assay, stem cell expression level assay, and cryopreservation damage assay, respectively, and the results were obtained. The test results will determine whether the revived muscle stem cells can be used subsequently.

[0009] The present invention has the following beneficial effects: (1) In this application, vitamin E succinate is used instead of traditional fat-soluble vitamin E. Its hydrophilic succinate group greatly improves its water solubility. It is stable in isotonic aqueous solution and does not precipitate. It has high bioavailability and its antioxidant efficiency is more than 3 times higher than that of the traditional form. Vitamin E succinate can specifically anchor to the inner mitochondrial membrane and work synergistically with magnesium sulfate heptahydrate to stabilize mitochondrial membrane function, forming a dual mitochondrial protection mechanism and improving the survival rate after resuscitation.

[0010] (2) In the present application, during cell resuscitation, mannan peptide can improve the cold resistance, anti-stress ability and oxidation resistance of muscle stem cells, and can also stabilize the cell membrane structure of muscle stem cells, so as to achieve the purpose of protecting muscle stem cells at low temperature; both gypenoside and portulaca oleracea polysaccharide can scavenge reactive oxygen species generated during the resuscitation of muscle stem cells, reduce oxidative stress, and reduce the loss of muscle stem cells; in addition, gypenoside and portulaca oleracea polysaccharide can also act as osmotic protectants to maintain cell membrane integrity, reduce water loss and ice crystal damage, and improve the survival rate of muscle stem cells.

[0011] (3) The washing solution in the present application is serum-free, protein-free, DMSO-free (English name: Dimethylsulfoxide), free of exogenous enzymes, with clear and simple components; all raw materials are of pharmaceutical grade or cell culture grade, which meets the basic requirements of GMP (Good Manufacturing Practice of Medical Products) for excipients of cell preparations, and is suitable for clinical application and industrial production.

[0012] (4) In the present application, the Pax7 / MyoD fluorescent double antibody pairing scheme is integrated into the muscle stem cell resuscitation quality assessment system, which can specifically determine the stemness maintenance status of muscle stem cells after resuscitation.

[0013] (5) The kit of the present application comprises an integrated closed-loop system constructed by the washing solution and specific assessment indicators, which achieves the synergistic technical effect that cannot be realized by a single component, solves the problem of disconnection between resuscitation treatment and effect assessment, and ensures the survival number of cells and the stability of cell function and stemness after resuscitation. Description of Drawings

[0014] Figure 1 is a comparison graph of cell survival rates of three groups of resuscitated muscle stem cells; Figure 2 is a comparison graph of cell proliferation rates of three groups of resuscitated muscle stem cells; Figure 3 is a comparison graph of the proportions of Pax7 / MyoD double positive cells in three groups of resuscitated muscle stem cells; Figure 4 is a comparison graph of ROS fluorescence intensities of three groups of resuscitated muscle stem cells; Figure 5 is a comparison graph of Caspase-3 activities of three groups of resuscitated muscle stem cells; Figure 6 is a comparison graph of LDH (English name: lactate dehydrogenase) release rates of three groups of resuscitated muscle stem cells. Specific Embodiments

[0015] In a first aspect, this application provides a muscle stem cell resuscitation and protective washing solution, which uses sterile deionized water as a solvent and comprises, according to a final concentration: 15-25 mM of 4-hydroxyethylpiperazine ethanesulfonic acid, 20-50 μg / mL of mannan peptide, 10-30 μg / mL of Gynostemma pentaphyllum polysaccharide, and 15-40 μg / mL of Portulaca oleracea polysaccharide. Sodium chloride 120-150mM, potassium chloride 4.0-7.0mM, trehalose 80-150mM, L-proline 4-10mM, vitamin E succinate 0.08-0.20mM, magnesium sulfate heptahydrate 0.5-1.5mM.

[0016] In this application, 4-hydroxyethylpiperazine ethanesulfonic acid is a physiological buffer regulator with excellent pH buffering capacity. During cell resuscitation, centrifugation, washing, and subsequent culture procedures, 4-hydroxyethylpiperazine ethanesulfonic acid can regulate tolerance to temperature changes, CO2 concentration fluctuations, and acid-base disturbances caused by reagent mixing, stabilizing the system pH within the physiologically suitable range of 7.30-7.40. Simultaneously, 4-hydroxyethylpiperazine ethanesulfonic acid does not participate in cell metabolism and has no cytotoxicity, avoiding pH drift caused by environmental gases in conventional carbonate buffers. This provides a constant and stable acid-base microenvironment throughout the muscle stem cell resuscitation process, reducing cell viability decline and stemness loss caused by pH stress.

[0017] Mannan peptides are off-white or slightly yellow amorphous powders that are readily soluble in water and contain no small molecule sugars such as monosaccharides or disaccharides, nor free amino acids. During cell resuscitation, water-soluble mannan peptides can enhance the cold resistance, stress resistance, and antioxidant capacity of muscle stem cells, while also stabilizing the cell membrane structure of muscle stem cells, thus protecting them at low temperatures. In the embodiments of this application, commercially available mannan peptide tablets or oral solutions are used.

[0018] Both Gynostemma pentaphyllum polysaccharide and Portulaca oleracea polysaccharide can scavenge reactive oxygen species during muscle stem cell resuscitation, reduce oxidative stress, and decrease muscle stem cell loss. In addition, Gynostemma pentaphyllum polysaccharide and Portulaca oleracea polysaccharide can also act as osmoprotectants, maintain cell membrane integrity, reduce water loss and ice crystal damage, and improve the survival rate of muscle stem cells.

[0019] In this embodiment, the preparation method of Gynostemma pentaphyllum polysaccharide includes: drying and pulverizing Gynostemma pentaphyllum, adding it to 95% ethanol for reflux extraction twice, filtering, evaporating and recovering the ethanol to obtain a defatted residue; mixing the defatted residue and deionized water at a mass-volume ratio of 1:10-1:20, and heating and extracting at 60-100℃ for 0.5-2h to obtain an extract; centrifuging the extract at 3000-10000r / min for 15min to obtain a supernatant; concentrating the supernatant under reduced pressure, allowing it to settle and precipitate at 4℃ with ethanol, washing the precipitate sequentially with 95% ethanol, anhydrous ethanol and acetone, and drying it under vacuum at 50-60℃ to obtain Gynostemma pentaphyllum polysaccharide.

[0020] In this embodiment, the preparation method of purslane polysaccharide includes: purslane is dried and pulverized, then added to petroleum ether and extracted by Soxhlet reflux at 30-60℃ for 3-4 hours to remove fat-soluble impurities, and dried to obtain defatted powder; the defatted powder and deionized water are mixed at a mass-to-volume ratio of 1:25-1:30, and ultrasonically extracted at an ultrasonic power of 200-300W and a temperature of 70-80℃ for 30-60 minutes to obtain an extract; the extract is concentrated under reduced pressure at 60℃ and -0.08MPa to 1 / 5-1 / 10 of its original volume, and then precipitated by standing with ethanol at 4℃ for 12-24 hours; after standing, the precipitate is centrifuged at 4000rpm / min for 10 minutes to obtain the precipitate. The precipitate was washed with chloroform / n-butanol at a volume ratio of 4:1, centrifuged, adsorbed by activated carbon, and dialyzed to remove molecular weights of 8000-14000 Da. It was then freeze-dried at -50°C to obtain purslane polysaccharide.

[0021] Sodium chloride and potassium chloride are used as isotonic buffer solutions. Trehalose can form an extracellular protective layer on the outside of muscle stem cells, stabilizing the phospholipid bilayer structure through hydrogen bond substitution and reducing membrane rupture caused by ice crystal damage. L-proline, as a natural osmotic regulator, can enter the interior of muscle stem cells, stabilizing the three-dimensional structure of proteins and thus preventing protein aggregation and precipitation caused by freeze-drying concentration. Mg in magnesium sulfate heptahydrate... 2+ Capable of stabilizing mitochondrial inner membrane Mg 2+ / ATPase channels prevent a sudden drop in mitochondrial membrane potential and maintain mitochondrial function.

[0022] Vitamin E succinate is a monosuccinate of α-tocopherol. Its molecular structure incorporates a hydrophilic succinic acid group, resulting in a solubility of at least 0.10 mM in isotonic aqueous solutions compared to ordinary fat-soluble vitamin E. This increased water solubility prevents precipitation in aqueous phases, allowing it to more effectively penetrate cell membranes and exert intracellular antioxidant effects. Simultaneously, it synergistically protects mitochondrial membrane function by inhibiting excessive leakage of the mitochondrial electron transport chain.

[0023] Thus, the above components together form an anti-damage system that provides isotonic buffering, extracellular protection, intracellular protein stabilization, antioxidant protection, and mitochondrial stabilization. This system synergistically reduces osmotic pressure damage, oxidative stress damage, and mitochondrial damage during the recovery process, maintaining the activity and stemness of muscle stem cells.

[0024] In this application, the osmotic pressure of the resuscitation and protective washing solution is 265-275 mOsm / kg. The pH of the resuscitation and protective washing solution is adjusted to 7.30-7.40 by using a 1M NaOH solution or a 1M HCl solution to suit the resuscitation and detection of muscle stem cells.

[0025] Preferred, the muscle stem cell resuscitation and protective washing solution in this application comprises, in a final concentration: 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 35 μg / mL mannan peptide, 20 μg / mL Gynostemma pentaphyllum polysaccharide, 25 μg / mL Portulaca oleracea polysaccharide, 135 mM sodium chloride, 5.4 mM potassium chloride, 120 mM trehalose, 6 mM L-proline, 0.12 mM vitamin E succinate, and 0.75 mM magnesium sulfate heptahydrate.

[0026] Secondly, this application provides a muscle stem cell resuscitation quality assessment kit, including the muscle stem cell resuscitation protective washing solution described in the first aspect.

[0027] In addition, the muscle stem cell resuscitation quality assessment kit also includes cell viability testing reagents, cell proliferation testing reagents, stem cell expression level testing reagents, and cryopreservation damage testing reagents.

[0028] In this application, the cell viability assay reagent is trypan blue staining solution or 7-AAD / Annexin-V flow cytometry assay reagent; the cell proliferation assay reagent is a CCK-8 assay kit or an EdU incorporation assay kit; the stem cell expression level assay reagent includes a paired Pax7 fluorescently labeled antibody and the MyoD fluorescently labeled antibody, used to specifically detect the stem cell expression level of muscle stem cells to assess the stem cell maintenance status after resuscitation. Specifically, the Pax7 fluorescently labeled antibody is a FITC-labeled anti-Pax7 monoclonal antibody, and the MyoD fluorescently labeled antibody is a PE-labeled anti-MyoD monoclonal antibody. The cryopreservation damage detection reagents include ROS (reactive oxygen species) detection reagents, Caspase-3 detection reagents, and LDH detection reagents. The ROS detection reagent includes the DCFH-DA (2',7'-Dichlorodihydrofluorescein diacetate) probe, the Caspase-3 detection reagent contains the fluorescent substrate Ac-DEVD-AMC, and the LDH detection reagent is used to detect the amount of LDH released from muscle stem cells.

[0029] Thirdly, this application provides a method for using a muscle stem cell resuscitation quality assessment kit, including: S01: Slowly add the thawed muscle stem cells to the muscle stem cell resuscitation and protection washing solution that has been brought to room temperature, mix well by pipetting, and centrifuge at 4°C and 1000 r / min for 5 min to obtain cell pellet.

[0030] Remove the muscle stem cell resuscitation and protection washing buffer from the 4°C refrigerated environment and place it at room temperature (25±2°C). Shake well before use, avoiding repeated freeze-thaw cycles. Remove the cryopreserved muscle stem cell tubes from the liquid nitrogen container and quickly place them in a 37°C constant temperature water bath. Shake the cryopreservation tubes rapidly to allow the muscle stem cells to thaw completely within 1-2 minutes. Slowly transfer the thawed muscle stem cells to a sterile centrifuge tube, add twice the volume of muscle stem cell resuscitation and protection washing buffer, gently pipette 3-5 times to mix, and centrifuge at 4°C, 1000 rpm for 5 minutes. Discard the supernatant to obtain the cell pellet.

[0031] S02: Add an equal volume of muscle stem cell resuscitation and protection washing solution to the cell pellet, gently resuspend by pipetting, centrifuge at 4℃ and 800r / min for 3min, discard the supernatant, and obtain the resuscitated muscle stem cells.

[0032] S03: Cell viability, cell proliferation, stem cell expression level, and cryopreservation damage level of revived muscle stem cells were detected using cell viability assay reagents, cell proliferation assay reagents, stem cell expression level assay reagents, and cryopreservation damage assay reagents, respectively, and the results were obtained.

[0033] The survival rate of muscle stem cells was detected using conventional staining or flow cytometry with cell viability assays; the proliferation rate of muscle stem cells was detected using CCK-8 assay or EdU assay with cell proliferation assays; the proportion of Pax7 / MyoD double-positive cells was detected using immunofluorescence staining with stem cell expression assays; and the intracellular ROS content, Caspase-3 activity, and extracellular LDH release of muscle stem cells were detected using ROS assays, Caspase-3 assays, and LDH assays, respectively, to comprehensively evaluate the degree of cellular oxidative and apoptotic damage.

[0034] S04: Determine whether the revived muscle stem cells should be used subsequently based on the test results.

[0035] When a batch of revived muscle stem cells simultaneously meets the following requirements: cell viability ≥85%, cell proliferation rate decrease ≤20% compared to pre-freezing, Pax7 / MyoD double positivity rate ≥80%, and ROS content, Caspase-3 activity, and LDH release all increase by no more than 30% compared to fresh muscle stem cells, the revived muscle stem cells meet the quality requirements for subsequent application and can be used. If any one of these requirements is not met, the revived muscle stem cells cannot be used.

[0036] The technical solution of the present invention will be further explained and illustrated below through specific embodiments. All reagents in this application are cell culture grade or pharmaceutical grade pure products, and all operations are performed in a sterile ultra-clean workbench to avoid contamination by other microorganisms. The ROS detection reagent, Caspase-3 detection reagent, and LDH detection reagent are all existing reagents. The mannan peptide is a commercially available mannan peptide tablet. The Gynostemma pentaphyllum polysaccharide and Portulaca oleracea polysaccharide can be commercially available Gynostemma pentaphyllum polysaccharide and Portulaca oleracea polysaccharide, or they can be prepared by the following methods respectively: The preparation method of Gynostemma pentaphyllum polysaccharide includes: 400g of dried and pulverized Gynostemma pentaphyllum is added to 2500mL of 95% ethanol and refluxed twice. After filtration, the ethanol is evaporated and recovered to obtain a defatted residue. The defatted residue and deionized water are mixed at a mass-volume ratio of 1:20 and heated at 80℃ for 1h to obtain an extract. The extract is centrifuged at 5000r / min for 15min to obtain a supernatant. The supernatant is concentrated under reduced pressure, allowed to stand in ethanol at 4℃ to precipitate, and then washed with 95% ethanol, anhydrous ethanol and acetone in sequence. After vacuum drying at 55℃, Gynostemma pentaphyllum polysaccharide is obtained.

[0037] The preparation method of purslane polysaccharide includes: 500g of dried and pulverized purslane is added to 3000mL of petroleum ether and extracted by Soxhlet reflux at 50℃ for 4h to remove fat-soluble impurities. After drying, a defatted powder is obtained. The defatted powder and deionized water are mixed at a mass-to-volume ratio of 1:25 and ultrasonically extracted at 75℃ for 50min to obtain an extract. The extract is concentrated under reduced pressure at 60℃ and -0.08MPa to 1 / 8 of its original volume and then allowed to stand in ethanol at 4℃ for 24h to precipitate. After standing, the precipitate is centrifuged at 4000rpm / min for 10min to obtain a precipitate. The precipitate is washed with chloroform / n-butanol at a volume ratio of 4:1, centrifuged, adsorbed by activated carbon, and dialyzed with a molecular weight cutoff of 10000Da. Finally, it is freeze-dried at -50℃ to obtain purslane polysaccharide.

[0038] Example 1 This application provides a muscle stem cell resuscitation and protective washing solution. The resuscitation and protective washing solution uses sterile deionized water as a solvent and comprises, according to the final concentration: 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 35 μg / mL mannan peptide, 20 μg / mL Gynostemma pentaphyllum polysaccharide, 25 μg / mL Portulaca oleracea polysaccharide, 135 mM sodium chloride, 5.4 mM potassium chloride, 120 mM trehalose, 6 mM L-proline, 0.12 mM vitamin E succinate, and 0.75 mM magnesium sulfate heptahydrate.

[0039] This application also provides a method for preparing a muscle stem cell resuscitation and protective washing solution, the method comprising: S101: Dissolve trehalose in a water bath at 45℃ to form a trehalose solution. Dissolve vitamin E succinate in anhydrous ethanol with a concentration ≤0.05% to form a vitamin E succinate solution. Add 900mL of sterile deionized water to a 1000mL sterile beaker. Under magnetic stirring at 150-180r / min, add the following components sequentially, based on a final volume of 1L: 4.766g 4-hydroxyethylpiperazine ethanesulfonic acid, 35mg mannan peptide, 20mg Gynostemma pentaphyllum polysaccharide, 25mg Portulaca oleracea polysaccharide, 7.894g sodium chloride, 0.403g potassium chloride, 45.40g trehalose solution, 0.691g L-proline, 0.064g vitamin E succinate solution, and 0.185g magnesium sulfate heptahydrate. The vitamin E succinate solution is added dropwise to the sterile beaker. After complete dissolution, bring the volume to 1L with sterile deionized water and continue stirring for 15 minutes to ensure no precipitation or turbidity, thus forming a mixed solution.

[0040] S102: After adjusting the pH of the mixture to 7.35 with 1M NaOH, use an osmometer to measure the osmotic pressure, ensuring it is 265-275 mOsm / kg. If the osmotic pressure is too high, add a small amount of deionized water; if the osmotic pressure is too low, add a small amount of NaCl for fine-tuning.

[0041] S103: After sterilization by filtration through a 0.22μm sterile filter membrane, the mixture is dispensed into 100mL vials, sealed with nitrogen, and labeled with the date and batch number. Shelf life is 6 months when refrigerated at 4℃ and 12 months when frozen at -20℃. After thawing, store at 4℃ and use within 72 hours; do not refrozen.

[0042] Example 2 This application provides a muscle stem cell resuscitation and protective washing solution. The resuscitation and protective washing solution uses sterile deionized water as a solvent and comprises, according to the final concentration: 15mM 4-hydroxyethylpiperazine ethanesulfonic acid, 35 μg / mL mannan peptide, 20 μg / mL Gynostemma pentaphyllum polysaccharide, 25 μg / mL Portulaca oleracea polysaccharide, 150mM sodium chloride, 4.0mM potassium chloride, 150mM trehalose, 4mM L-proline, 0.08mM vitamin E succinate, and 1.5mM magnesium sulfate heptahydrate.

[0043] This application also provides a method for preparing a muscle stem cell resuscitation and protective washing solution, which is the same as in Example 1.

[0044] Example 3 This application provides a muscle stem cell resuscitation and protective washing solution. The resuscitation and protective washing solution uses sterile deionized water as a solvent and comprises, according to the final concentration: 25 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 35 μg / mL mannan peptide, 20 μg / mL Gynostemma pentaphyllum polysaccharide, 25 μg / mL Portulaca oleracea polysaccharide, 150 mM sodium chloride, 4.0 mM potassium chloride, 80 mM trehalose, 10 mM L-proline, 0.20 mM vitamin E succinate, and 0.5 mM magnesium sulfate heptahydrate.

[0045] This application also provides a method for preparing a muscle stem cell resuscitation and protective washing solution, which is the same as in Example 1.

[0046] Example 4 This application provides a muscle stem cell resuscitation quality assessment kit, comprising one bottle of muscle stem cell resuscitation protective washing solution as described in Example 1, 10 mL of 0.4% trypan blue staining solution, 1 mL of CCK-8 assay kit, 100 μL / tube of 1 mg / mL FITC-labeled anti-human / mouse Pax7 monoclonal antibody, 100 μL / tube of 1 mg / mL PE-labeled anti-human / mouse MyoD monoclonal antibody, ROS assay reagent, Caspase-3 assay reagent, and LDH assay reagent. The kit should be stored at 4°C protected from light, and the fluorescent antibody should be stored at -20°C protected from light. Before use, thaw at room temperature and mix thoroughly.

[0047] Example 5 This application provides a muscle stem cell resuscitation quality assessment kit, comprising one vial of the muscle stem cell resuscitation protective washing solution from Example 2, a 7-AAD / Annexin-V flow cytometry assay kit, a 1 mL CCK-8 assay kit, 100 μL / tube, 1 mg / mL FITC-labeled anti-human / mouse Pax7 monoclonal antibody, 100 μL / tube, 1 mg / mL PE-labeled anti-human / mouse MyoD monoclonal antibody, ROS assay reagent, Caspase-3 assay reagent, and LDH assay reagent. The kit should be stored at 4°C protected from light, and the fluorescent antibody should be stored at -20°C protected from light. Before use, thaw at room temperature and mix thoroughly.

[0048] Example 6 This application provides a muscle stem cell resuscitation quality assessment kit, comprising one bottle of muscle stem cell resuscitation protective washing solution as described in Example 3, 10 mL of 0.4% trypan blue staining solution, an EdU incorporation detection kit, 100 μL / tube of 1 mg / mL FITC-labeled anti-human / mouse Pax7 monoclonal antibody, 100 μL / tube of 1 mg / mL PE-labeled anti-human / mouse MyoD monoclonal antibody, ROS detection reagent, Caspase-3 detection reagent, and LDH detection reagent. The kit should be stored at 4°C protected from light, and the fluorescent antibody should be stored at -20°C protected from light. Before use, thaw at room temperature and mix thoroughly.

[0049] Example 7 This application's embodiments utilize the muscle stem cell resuscitation quality assessment kit, PBS solution (phosphate buffer saline), and Ca-free solution from Example 4, respectively. 2+ / Mg 2+ The use of HBSS (Hank's Balanced Salt Solution) for the resuscitation, protection, and quality assessment of cryopreserved human muscle stem cells is detailed below: 1. Experimental Materials Human primary muscle stem cells with Pax7 / MyoD double-positive purity ≥75% were cryopreserved under the following conditions: complete culture medium containing 10% DMSO, stored in liquid nitrogen at -196°C for 3 months. Group 1: isotonic ordinary PBS solution at pH 7.4; Group 2: HBSS; Group 3: muscle stem cell resuscitation quality assessment kit from Example 2. Each experiment was repeated in triplicate, and the mean ± standard deviation was taken.

[0050] 2. Recovery Preparation Remove the muscle stem cell resuscitation and protective washing solution from the muscle stem cell resuscitation quality assessment kit in Example 2 from 4°C, let it stand at room temperature for 30 minutes, and gently shake well before use. Groups 1 and 2 were treated similarly.

[0051] 3. Cell thawing Remove the cryopreservation tubes containing muscle stem cells from the liquid nitrogen tank, quickly place them in a 37°C constant temperature water bath, shake rapidly for 1.5 minutes until completely thawed, and divide them into 3 groups.

[0052] 4. Resuscitation and Washing The thawed muscle stem cells from the three groups were transferred into three 15 mL sterile centrifuge tubes. 2 mL of PBS solution was added to group 1, 2 mL of HBSS to group 2, and 2 mL of the muscle stem cell resuscitation and protection washing solution from Example 2 to group 3. Each tube was gently pipetted four times, centrifuged at 4°C and 1000 rpm for 5 min, and the supernatant was discarded. Group 1 was resuspended in 1 mL of PBS solution, Group 2 in 1 mL of HBSS, and Group 3 in 1 mL of the muscle stem cell resuscitation and protection washing solution from Example 2. Each tube was centrifuged at 4°C and 800 rpm for 3 min, and the supernatant was discarded, yielding the resuscitated muscle stem cells.

[0053] 5. Quality Assessment and Results 5.1 Cell viability Three groups of 10 μL each of resuscitated muscle stem cells were mixed with 10 μL of trypan blue staining solution, counted under a microscope, and the survival rate was calculated. Figure 1 From the appendix Figure 1 It can be seen that the survival rates of revived muscle stem cells in groups 1, 2 and 3 were 75.3±4.2%, 80.5±3.5% and 95.2±2.1%, respectively, and the survival rate of group 3 was significantly higher than that of groups 1 and 2.

[0054] 5.2 Cell proliferation rate Three groups of revived muscle stem cells were seeded into 96-well plates, with 1 × 10⁶ cells per well. 4 Cells were cultured for 24 hours, and then CCK-8 reagent was added. After incubation at 37°C for 2 hours, the absorbance at 450 nm was measured using a microplate reader to obtain the attached cells. Figure 2From the appendix Figure 2 It can be seen that the cell proliferation rates of revived muscle stem cells in groups 1, 2 and 3 were 59.0±3.7%, 74.8±2.3% and 92.5±3.8%, respectively, and the cell proliferation rate of group 3 was significantly higher than that of groups 1 and 2.

[0055] 5.3 Stem expression level Three groups of revived muscle stem cells were seeded onto crawling slides and cultured for 48 hours. After fixation and permeabilization, Pax7 and MyoD fluorescently labeled antibodies were added, and the cells were incubated overnight. The cells were observed under a fluorescence microscope, and the proportion of Pax7 / MyoD double-positive cells was calculated. Figure 3 From the appendix Figure 3 It can be seen that the Pax7 / MyoD double positivity rates of resuscitated muscle stem cells in groups 1, 2 and 3 were 61.4±5.1%, 71.1±4.5% and 88.3±3.2%, respectively, and the Pax7 / MyoD double positivity rate of group 3 was significantly higher than that of groups 1 and 2.

[0056] 5.4 Level of cryopreservation damage The intracellular ROS content, enzyme activity, and extracellular LDH release of three groups of resuscitated muscle stem cells were detected using ROS, Caspase-3, and LDH assays, respectively. Figure 4-6 From the appendix Figure 4-6 As can be seen, Group 1 exhibited the highest ROS fluorescence intensity, Caspase-3 activity, and LDH release rate, at 3200±150, 2500±130, and 25.1±3.1%, respectively. Group 2 showed a decrease in all indicators, at 2500±120, 1800±110, and 18.5±2.3%, respectively. Group 3, with ROS fluorescence intensity, Caspase-3 activity, and LDH release rate of 1250±85, 850±62, and 8.2±1.5%, was significantly lower than Groups 1 and 2. This indicates that the kit provided in this application can effectively reduce oxidative stress and apoptotic damage to cells during cryopreservation and thawing.

[0057] The above results demonstrate that the kit provided in this application can effectively achieve efficient resuscitation and protection of muscle stem cells, with significant synergistic effects among its components. The multi-dimensional evaluation indicators can accurately quantify the quality of stem cell resuscitation, and each detection indicator forms a stable functional association and synergistic adaptation relationship with the washing and protection effect.

[0058] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A muscle stem cell resuscitation and protection washing solution, characterized in that, The resuscitation and protective washing solution uses sterile deionized water as a solvent and comprises, at a final concentration: 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 35 μg / mL mannan peptide, 20 μg / mL Gynostemma pentaphyllum polysaccharide, 25 μg / mL Portulaca oleracea polysaccharide, 135 mM sodium chloride, 5.4 mM potassium chloride, 120 mM trehalose, 6 mM L-proline, 0.12 mM vitamin E succinate, and 0.75 mM magnesium sulfate heptahydrate. The resuscitation and protective washing solution is a serum-free, protein-free, DMSO-free, exogenous enzyme-free washing solution with a pH of 7.30-7.40 and an osmotic pressure of 265-275 mOsm / kg.

2. A muscle stem cell resuscitation quality assessment kit, characterized in that, Includes the muscle stem cell resuscitation and protection washing solution as described in claim 1.

3. The muscle stem cell resuscitation quality assessment kit according to claim 2, characterized in that, It also includes reagents for detecting cell viability, cell proliferation, stem cell expression levels, and cryopreservation damage.

4. The muscle stem cell resuscitation quality assessment kit according to claim 3, characterized in that, The cell viability detection reagent is trypan blue staining solution or 7-AAD / Annexin-V flow cytometry reagent.

5. The muscle stem cell resuscitation quality assessment kit according to claim 3, characterized in that, The cell proliferation rate detection reagent is either a CCK-8 detection kit or an EdU incorporation detection kit.

6. The muscle stem cell resuscitation quality assessment kit according to claim 3, characterized in that, The reagent for detecting the dry expression level includes a pair of Pax7 fluorescently labeled antibody and a MyoD fluorescently labeled antibody.

7. The muscle stem cell resuscitation quality assessment kit according to claim 3, characterized in that, The cryopreservation damage detection reagents include ROS detection reagents, Caspase-3 detection reagents, and LDH detection reagents.

8. The method of using the muscle stem cell resuscitation quality assessment kit according to any one of claims 3-7, characterized in that, include: The thawed muscle stem cells were slowly added to the muscle stem cell resuscitation and protection washing solution that had been brought to room temperature. After mixing by pipetting, the mixture was centrifuged at 4°C and 1000 r / min for 5 min to obtain cell pellet. The cell pellet was added to fresh muscle stem cell resuscitation and protective washing solution, resuspended by pipetting, and centrifuged at 4°C and 800 r / min for 3 min to obtain resuscitated muscle stem cells. The cell viability, cell proliferation, stem cell expression level, and cryopreservation damage level of the revived muscle stem cells were detected using cell viability assay, cell proliferation assay, stem cell expression level assay, and cryopreservation damage assay, respectively, and the results were obtained. The test results will determine whether the revived muscle stem cells can be used subsequently.

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