Stem cell exosome preparation for xerophthalmia or eye ulcer and preparation method of stem cell exosome preparation

Abstract

The invention discloses a stem cell exosome preparation for xerophthalmia or eye ulcer and a preparation method of the stem cell exosome preparation, and belongs to the technical field of biological medicine. The method comprises the following steps: carrying out low-intensity ultrasonic pulse treatment on the mesenchymal stem cells by adopting a pretreatment culture medium containing interferon-gamma and tumor necrosis factor-alpha; removing the pretreated culture medium, replacing the pretreated culture medium with a serum-free culture medium for continuous culture, collecting a culture solution, performing differential centrifugation, and collecting supernate; concentrating the supernate by using a tangential flow filtration system to obtain a concentrated solution; and loading the sample to a size exclusion chromatographic column for purification, and collecting an eluent to obtain the stem cell exosome. The invention discloses a stem cell exosome preparation for xerophthalmia or eye ulcer and a preparation method of the stem cell exosome preparation. A novel biological preparation which can be produced on a large scale and is safe and effective is provided for clinical treatment of xerophthalmia and corneal ulcer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a stem cell exosome preparation for dry eye or eye ulcer and a preparation method thereof. BACKGROUND

[0002] Exosomes are nanoscale vesicles secreted by cells, which can carry proteins, lipids, miRNAs, mRNAs and other nucleic acid bioactive molecules, and play a key role in cell-to-cell signal transmission, physiological function regulation and disease development. Among them, stem cell-derived exosomes have similar biological functions to stem cells, and have become the focus of research in the fields of regenerative medicine and disease treatment.

[0003] In particular, in the treatment of ophthalmic diseases, stem cell exosomes have broad prospects. Dry eye, corneal ulcer and other diseases are often accompanied by ocular surface tissue damage and inflammation imbalance, and their good biocompatibility, tissue repair ability and anti-inflammatory properties are expected to become a new treatment for such diseases, providing a new path for improving patients' vision and ocular surface health.

[0004] However, the existing technology seriously restricts the clinical transformation of stem cell exosomes. First, the contradiction between yield and activity is prominent. Conventional two-dimensional culture is difficult to achieve efficient proliferation of stem cells, and the combination of traditional ultracentrifugation extraction not only has low yield and cannot meet the clinical demand, but also easily leads to the loss or degradation of active ingredients such as specific miRNA and functional proteins in exosomes, reducing the treatment effect. Second, the purity and impurity problem is significant. Ultracentrifugation is easy to co-precipitate exosomes with protein polymers, serum impurities, cell debris and other substances, and residual impurities may cause immune response, affecting the safety of use. Third, the function is single. The function of natural stem cell exosomes is fixed, and it cannot strengthen the function for specific diseases such as tear secretion deficiency of dry eye and persistent inflammation of corneal ulcer, and the adaptability and effectiveness are limited. SUMMARY

[0005] The present application aims to provide a stem cell exosome preparation for dry eye or eye ulcer and a preparation method thereof, which provides a new type of biological preparation that can be produced on a large scale and is safe and effective for the clinical treatment of dry eye and corneal ulcer.

[0006] To solve the above technical problems, the technical solution adopted by the present application is as follows: A preparation method of stem cell exosomes, comprising the following steps: S1, low-intensity ultrasonic pulse treatment is performed on mesenchymal stem cells using a pretreatment medium containing interferon-gamma and tumor necrosis factor-alpha; S2, the pretreatment medium is removed and replaced with a serum-free medium for continuous culture, and the culture solution is collected, S3, differential centrifugation is performed on the culture solution collected in S2, and the supernatant is collected; S4. Concentrate the supernatant collected in S3 using a tangential flow filtration system to obtain a concentrated solution; S5. The concentrated solution obtained in S4 is loaded onto a size exclusion chromatography column for purification, and the eluent is collected to obtain stem cell exosomes.

[0007] Preferably, in S1, the concentration of interferon-γ is 20-100 ng / mL, and the concentration of tumor necrosis factor-α is 10-50 ng / mL.

[0008] Preferably, in S1, the intensity of the low-intensity ultrasonic pulse treatment is 0.3-0.8 W / cm. 2 The frequency is 200-500kHz, and the pulse mode is: working time is 2-5s, interval is 6-7s, and continuous processing time is 30-120min.

[0009] Preferably, in S3, the differential centrifugation includes: centrifugation at 300-500×g for 8-12 minutes at 4°C; centrifugation at 2000-3000×g for 15-25 minutes at 4°C; and centrifugation at 10000-12000×g for 30-40 minutes at 4°C.

[0010] Preferably, in S4, the tangential flow filtration system uses a hollow fiber column membrane with a molecular weight cutoff of 100-300kDa, a flow rate of 50-60mL / min, and a transmembrane pressure of 18-20psi.

[0011] The present invention also provides stem cell exosomes prepared by the aforementioned preparation method.

[0012] The present invention also provides the use of the said stem cell exosomes in the preparation of formulations for treating dry eye syndrome or corneal ulcers.

[0013] The present invention also provides a formulation for treating dry eye syndrome, comprising the said stem cell exosomes and a pharmaceutically acceptable carrier.

[0014] The present invention also provides a formulation for corneal ulcers, comprising the said stem cell exosomes and a pharmaceutically acceptable carrier.

[0015] Preferably, the pharmaceutically acceptable carrier includes one or more of the following: solvent, dispersant, suspending agent, surfactant, isotonic agent, thickener, preservative, solid binder, or lubricant. Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses a stem cell exosome preparation for dry eye syndrome or ocular ulcers and its preparation method. Pretreatment with interferon-γ and tumor necrosis factor-α activates the paracrine pathway of mesenchymal stem cells, increasing exosome release while selectively enriching anti-inflammatory and ocular surface repair-related functional molecules. Animal experiments have demonstrated that the exosomes prepared in this invention can restore tear secretion in BAC-induced dry eye model mice and reduce the content of ocular surface inflammatory factors. This provides a novel, safe, and effective biological agent that can be mass-produced for the clinical treatment of dry eye syndrome and corneal ulcers, possessing significant clinical translational value.

[0016] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0017] The technical solution of the present invention will be further illustrated by the following embodiments.

[0018] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0019] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0020] Pretreatment medium: DMEM basal medium, 10% (v / v) exosome-free FBS treated by ultracentrifugation, 50 ng / mL interferon-γ (IFN-γ), 25 ng / mL tumor necrosis factor-α (TNF-α), 2 mM L-glutamine, 1% (v / v) non-essential amino acids, 1% (v / v) penicillin-streptomycin solution; Serum-free culture medium: DMEM basal medium, 1% (v / v) ITS (insulin-transferrin-selenium) additive, 0.5% (w / v) recombinant human serum albumin (rHSA), 2 mM GlutaMAX™, 10 mM HEPES, 1% (v / v) penicillin-streptomycin solution.

[0021] Example 1 Human umbilical cord tissue was collected, excluding the blood vessels of the umbilical cord membrane, and only Wharton's jelly tissue was preserved. This tissue was then cut into 1-2 mm pieces. 3 Small pieces are preprocessed.

[0022] Primary cell isolation and culture Pretreated Wharton's jelly fragments were seeded into DMEM medium containing 10% fetal bovine serum, 2 mM L-glutamine, and 1% penicillin-streptomycin, and cultured in a 37°C, 5% CO2 incubator. After 7 days of culture, spindle-shaped mesenchymal stem cells migrated from around the Wharton's jelly fragments. When the cell confluence reached 85%, the cells were collected by trypsin digestion, which yielded primary UC-MSCs.

[0023] Expansion to the 4th generation Primary UC-MSCs were seeded into new culture flasks at a ratio of 1:3 and passaged using the same culture medium as the primary culture. The medium was changed every 3 days, and the next passage was performed when the cell confluence reached more than 85%. When passaged to the 4th generation, the cells exhibited uniform morphology, typical spindle shape, no obvious differentiation or senescence, stable proliferative activity, no abnormal genetic background, and surface markers that met the standards for mesenchymal stem cells. At this point, they could be used as donor cells for exosome preparation.

[0024] A method for preparing stem cell exosomes includes the following steps: S1. Take P4 generation UC-MSCs with good growth and 80% confluence, discard the old culture medium, gently wash the cells twice with pre-warmed PBS buffer (pH=7.4), add pretreatment culture medium, place the culture flask on the flat probe of an ultrasound therapy instrument, and perform low-intensity ultrasound pulse treatment (intensity 0.5W / cm). 2 The frequency was 300kHz, pulse mode: 3 seconds working, 6 seconds interval, total processing time was 60 minutes). The treatment was carried out in a 37℃, 5%CO2 incubator to ensure a stable cell environment. S2. After sonication, carefully aspirate all the pretreatment culture medium and gently rinse the cell surface three times with preheated PBS buffer (pH=7.4) to thoroughly remove serum and cytokines. Replace with serum-free collection medium and continue culturing in a 37°C, 5% CO2 incubator for 48 hours. After culturing, collect the cell culture supernatant with a pipette. To remove a small number of detached cells, centrifuge the collected supernatant at 4°C, 300×g for 10 min and collect the supernatant to obtain the culture medium. S3. Centrifuge the culture medium collected in S2 at a differential speed, specifically: centrifuge at 300×g for 10 min at 4℃; centrifuge at 2000×g for 20 min at 4℃; centrifuge at 10000×g for 35 min at 4℃, and collect the supernatant. S4. Load the supernatant obtained in S3 into a tangential flow filtration system, use a hollow fiber column membrane with a molecular weight cutoff of 100kDa, set the flow rate to 55mL / min and the transmembrane pressure to 19psi, start the system to circulate and concentrate until the liquid volume is concentrated to about 15mL. S5. The concentrated solution obtained in S4 is loaded onto a size exclusion chromatography column for purification, specifically as follows: Using the AKTA pure purification system, all concentrates were filtered through a 0.22 μm filter membrane before loading. The chromatographic column was a Lonex Sepharose (XK 16 / 70); Equilibration: Equilibrate the column with 2-3 column volumes of PBS (pH=7.4) until the baseline is stable; Sample loading: Load the concentrate obtained from S4 at a flow rate of 1.0 mL / min; Elution: Isocratic elution was performed with PBS at a flow rate of 1.0 mL / min; Collection: Exosomes are collected based on the ultraviolet absorption peak (280 nm), and typically concentrate in the exclusion volume (V). o In the first main peak near the ) , the collection is concentrated in the exclusion volume (V o The eluent from the first main peak near the ) was collected to obtain stem cell exosomes.

[0025] Example 2 The preparation method is the same as in Example 1, except that the concentration of IFN-γ in the pretreatment culture medium is 20 ng / mL and the concentration of TNF-α is 50 ng / mL.

[0026] Example 3 The preparation method is the same as in Example 1, except that the concentration of IFN-γ in the pretreatment culture medium is 100 ng / mL and the concentration of TNF-α is 10 ng / mL.

[0027] Example 4 The preparation method is the same as in Example 1, except that low-intensity ultrasonic pulse treatment is used, with an intensity of 0.3 W / cm. 2 The frequency is 500kHz, and the pulse mode is 5 seconds for operation and 7 seconds for interval, with a total processing time of 80 minutes.

[0028] Comparative Example 1 The preparation method is the same as in Example 1, except that IFN-γ and TNF-α are not added to the pretreatment culture medium.

[0029] Comparative Example 2 The preparation method is the same as in Example 2, except that low-intensity ultrasonic pulse treatment is not performed.

[0030] The effects of stem cell exosomes in the above-described embodiments and comparative examples were verified through the following experiments.

[0031] Fifty-six healthy male C57BL / 6 mice, weighing 18-22g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were kept at 25±1℃ under a 12-hour light / dark cycle, with free access to water and food. They were fed a standard pelleted diet, typically consisting of various grains or cereal products made from soybean meal, corn flour, fish meal, and animal by-products. All experimental protocols were performed in accordance with the "Principles of Laboratory Animal Care and Research Use" (1988).

[0032] Fifty-six male C57BL / 6 mice that had been acclimatized for one week were weighed and recorded. They were randomly divided into seven groups of eight mice each: a control group, an example group, and a comparative group. Each group was treated with either Control or benzalkonium chloride (BAC). BAC solution was instilled into the eyes of mice to establish the model. The control group did not establish the model. The benzalkonium chloride (BAC) treatment was performed by instilling 0.1% BAC solution into each mouse twice a day for 7 days. The control group was treated with the same volume of sterile water.

[0033] To ensure the reliability of experimental results, stem cell exosome eye drops were prepared during the experimental interval between modeling and treatment. A sterile centrifuge tube was filled with 900 μL of sterile, endotoxin-free PBS solution (pH 7.4), and 100% glycerol was added to achieve a final concentration of 0.15%. 2 mg of sodium hyaluronate powder was then added, and the solution was gently vortexed at room temperature to completely dissolve the exosomes, forming a mixture for later use. The stem cell exosomes were then dissolved in the above solvent to obtain a clear solution with a concentration of 1.0 × 10⁻⁶. 9 μg / mL.

[0034] After modeling, mice were allowed free access to food and water. After 7 days of modeling, treatment began. During the treatment period, 2 μL of the corresponding drug solution or solvent was instilled into each eye three times a day for 14 days. The same volume of the above mixture was also instilled into the control group.

[0035] After the experiment, the tear secretion of mice in each group was measured. The specific experimental protocol is as follows: Remove the mouse from its cage beforehand and allow it to acclimatize on the experimental table for a few minutes. Gently restrain the mouse to avoid excessive struggling and stress-induced tearing. Use sterile forceps to remove a phenol red cotton thread. Hold the unstained end of the thread. Gently pull back the mouse's lower eyelid and place the bent end (stained end) of the thread into the conjunctival fornix in the outer third of the lower eyelid, ensuring it is in contact with the surface of the eyeball. Start a timer simultaneously and time precisely 60 seconds. During this time, keep the mouse's head stable to prevent it from scratching with its front paws. After 60 seconds, quickly and gently remove the thread. Immediately use calipers to measure the length (mm) of the reddened portion of the thread.

[0036] Record the data, with each eye measured individually and recorded as left and right eye values. The results are shown in Table 1 below. The groups are divided into: Control group; stem cell exosome group (stem cell exosomes provided in each embodiment); modeling group: benzalkonium chloride (BAC) group; and BAC + stem cell exosome group (stem cell exosomes provided in each embodiment).

[0037] Table 1. Tear secretion in mice of each group

[0038] As shown in Table 1, after BAC modeling and treatment, the tear secretion of all example groups was significantly higher than that of the benzalkonium chloride (BAC) group, and close to the level of their respective control groups. This indicates that the stem cell exosomes prepared according to the present invention have significant therapeutic effects on dry eye syndrome.

[0039] The ELISA kit was used to measure inflammatory factors in mouse ocular cells. The specific experimental protocol is as follows: After the above experiments, mice were immediately euthanized, and the eyeballs were carefully and completely removed using fine ophthalmic scissors and forceps. Under a dissecting microscope, corneal tissue was excised along the limbus (the junction of the cornea and sclera) using microsurgical scissors. The corneal / conjunctival tissue was homogenized in pre-cooled PBS, then centrifuged at high speed (12000 rpm, 10 min, 4℃). The supernatant was collected for analysis, strictly following the kit instructions. The results are shown in Table 2.

[0040] Table 2. Levels of inflammatory factors in mice of each group

[0041] As shown in Table 2, after BAC modeling and treatment, the levels of inflammatory factors in all example groups were much lower than those in the benzalkonium chloride (BAC) group. This indicates that the stem cell exosomes prepared according to the present invention have strong anti-inflammatory capabilities and can effectively inhibit BAC-induced inflammation.

[0042] 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 them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing stem cell exosomes, characterized in that, Includes the following steps: S1. Mesenchymal stem cells were treated with low-intensity ultrasound pulses using a pretreatment medium containing interferon-γ and tumor necrosis factor-α. S2. Remove the pretreated culture medium, replace it with serum-free culture medium and continue culturing, then collect the culture medium. S3. Centrifuge the culture medium collected in S2 at a differential speed and collect the supernatant; S4. Concentrate the supernatant collected in S3 using a tangential flow filtration system to obtain a concentrated solution; S5. The concentrated solution obtained in S4 is loaded onto a size exclusion chromatography column for purification, and the eluent is collected to obtain stem cell exosomes.

2. The preparation method according to claim 1, characterized in that, In S1, the concentration of interferon-γ is 20-100 ng / mL, and the concentration of tumor necrosis factor-α is 10-50 ng / mL.

3. The preparation method according to claim 1, characterized in that, In S1, the intensity of the low-intensity ultrasonic pulse treatment is 0.3-0.8 W / cm². 2 The frequency is 200-500kHz, and the pulse mode is: working time is 2-5s, interval is 6-7s, and continuous processing time is 30-120min.

4. The preparation method according to claim 1, characterized in that, In S3, the differential centrifugation includes: centrifugation at 300-500×g for 8-12 minutes at 4°C; centrifugation at 2000-3000×g for 15-25 minutes at 4°C; and centrifugation at 10000-12000×g for 30-40 minutes at 4°C.

5. The preparation method according to claim 1, characterized in that, In S4, the tangential flow filtration system uses a hollow fiber column membrane with a molecular weight cutoff of 100-300kDa, a flow rate of 50-60mL / min, and a transmembrane pressure of 18-20psi.

6. Stem cell exosomes prepared by the preparation method according to any one of claims 1-5.

7. The use of stem cell exosomes as described in claim 6 in the preparation of formulations for treating dry eye syndrome or corneal ulcers.

8. A preparation for treating dry eye syndrome, characterized in that, Includes the stem cell exosomes of claim 6 and pharmaceutically acceptable carriers.

9. A preparation for corneal ulcers, characterized in that, Includes the stem cell exosomes of claim 6 and pharmaceutically acceptable carriers.

10. The formulation according to claim 8 or 9, characterized in that, The pharmaceutically acceptable carriers include one or more of the following: solvents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, preservatives, solid binders, and lubricants.

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