Laminarin extracellular vesicles, and preparation method and application thereof

Abstract

This invention relates to the field of bioengineering technology, specifically to an extracellular vesicle from kelp, its preparation method, and its applications. This invention provides a method for efficiently isolating extracellular vesicles with specific biological activities from kelp, and an application scheme for these vesicles to promote the proliferation, migration, and maintenance of stemness of large yellow croaker muscle stem cells under low serum conditions. This overcomes the problems of high cost and limited functionality of fetal bovine serum as a substitute in existing cell-cultured meat technologies, providing a novel culture medium supplement component that is stable in origin, low in cost, and has a clear effect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to an extracellular vesicle of kelp, its preparation method, and its application. Background Technology

[0002] Kelp, as one of my country's major seaweed species and a representative of both food and medicine, is rich in polysaccharides, polyphenols, amino acids, proteins, vitamins, mannitol, and other nutrients beneficial to cell growth regulation. Ancient Chinese herbal medicine texts and modern scientific research have also shown that kelp possesses anti-inflammatory, antioxidant, and antibacterial effects. Extracellular vesicles can inherit the biological functions of the parent plant, and due to the protection of the lipid bilayer, they can more fully and stably exert the activity of their contents. Therefore, the development of kelp extracellular vesicles has promising application prospects.

[0003] Cell-cultured meat is a highly efficient and precise production of animal-derived foods through controlled in vitro cell culture, which can greatly improve resource utilization and material conversion rates. However, large-scale cell culture still faces the cost challenge of expensive fetal bovine serum. Therefore, developing low-cost culture media is a necessary condition for the industrial production of cell-cultured meat. However, developing novel, naturally derived serum alternatives with high proliferative capacity, safety, controllability, and sustainable supply is essential to meeting the dual requirements of specificity and stability of culture media components for large-scale cell culture production.

[0004] Food-derived extracellular vesicles naturally contain nutrients such as proteins, lipids, nucleic acids, and secondary metabolites found in cell culture media. They also possess advantages such as wide availability, low cost, and good biocompatibility, making them a potential supplement to natural low-serum culture media. Currently, technologies related to plant-derived extracellular vesicles or cell proliferation promotion include: Chinese Patent CN120424865A, which discloses a method for preparing and applying stem cell exosome vesicles induced by functional components of traditional Chinese medicine. Using turmeric and / or Polygonum cuspidatum as exosome inducers, this method can induce stem cell proliferation and enhanced exosome secretion, while also increasing exosome yield and loading of bioactive molecules, significantly improving the bioavailability of exosomes in diabetes treatment. Chinese Patent CN118773111A discloses a method for obtaining exosomes and its application, designed for large-scale production of highly therapeutic extracellular vesicles (EVs). Compared to traditional two-dimensional culture techniques, MSCs cultured in 3D environments secrete higher levels of extracellular vesicles (EVs) and exhibit stronger abilities to promote the proliferation, migration, and anti-aging of chondrocytes and human dermal fibroblasts. They also promote the polarization of macrophages from pro-inflammatory to anti-inflammatory morphologies. However, none of the aforementioned techniques have disclosed the potential of dietary extracellular vesicles as serum substitutes for muscle cell culture. Therefore, discovering dietary extracellular vesicles capable of achieving cell proliferation and stemness maintenance during large-scale cell expansion is of great significance. Summary of the Invention

[0005] The technical problem to be solved by this invention is that there is currently limited research on the regulatory mechanism of food-derived extracellular vesicles as serum substitutes for muscle cell culture, and it is still unclear whether extracellular vesicles with specific biological activities that are efficiently isolated from kelp can be used as serum substitutes.

[0006] To address the problems of existing technologies, this invention provides a method for efficiently isolating extracellular vesicles with specific biological activities from kelp, and an application scheme for these vesicles to promote the proliferation, migration, and stemness maintenance of large yellow croaker muscle stem cells under low serum conditions. This overcomes the problems of high cost of fetal bovine serum and limited functionality of alternatives in existing cell-cultured meat technologies, providing a novel culture medium supplement component that is stable in origin, low in cost, and has a clear effect.

[0007] To achieve the above objectives, the present invention provides a method for preparing extracellular vesicles of kelp, comprising the following steps:

[0008] (1) Raw material pretreatment: Wash and crush the dried kelp, mix it with phosphate buffer solution, extract the kelp juice, and form a suspension;

[0009] (2) Enzymatic hydrolysis: Add a complex enzyme solution of alginate lyase, cellulase and pectinase to the above suspension, and carry out a mild enzymatic hydrolysis reaction under the conditions of 25-45℃, pH 6.0-8.0 and hydrolysis time of 6-12 h, aiming to efficiently lyse the plant cell wall and release intracellular vesicles, while protecting the integrity of the vesicle structure to the maximum extent.

[0010] (3) Primary separation: After the enzymatic hydrolysis reaction is completed, the supernatant is collected after differential centrifugation; large tissue fragments and incompletely digested residues are removed by a series of low-speed centrifugations, and the supernatant containing vesicles is collected.

[0011] (4) Stepwise filtration: The supernatant was filtered sequentially through filter membranes with pore sizes of 0.8 μm, 0.45 μm, and 0.22 μm, and the filtrate was collected. The 0.8 μm filter membrane was mainly used to filter out large solid residues (such as undigested kelp tissue fragments, cell wall debris, and colloidal aggregates) that were not completely degraded after enzymatic hydrolysis of the kelp homogenate. During the collection of the supernatant after centrifugation, some of the large solid residues in the precipitate were collected along with the supernatant. The 0.8 μm filter membrane first intercepted the largest impurities, which could prevent them from clogging the subsequent smaller pore size filter membranes, thus reducing the burden on the subsequent fine filtration and improving the overall filtration efficiency. The 0.45 μm filter membrane is primarily used to filter submicron particles that can pass through a 0.8 μm filter membrane, such as alginate, oligosaccharide aggregates from incomplete cellulosic hydrolysis, and colloidal particles from pectin degradation. The 0.45 μm membrane further clarifies the enzymatic hydrolysis supernatant, removes colloidal impurities that cause filtrate viscosity, improves filtrate flowability, and provides better feed conditions for subsequent 0.22 μm membrane filtration. The 0.22 μm membrane is mainly used to filter residual enzyme protein aggregates and most microorganisms such as bacteria and fungal spores. Furthermore, the supernatant filtered through the 0.22 μm membrane undergoes sterilization filtration, ensuring the filtrate is sterile and preventing microbial growth and sample contamination during subsequent storage or purification. The advantage of passing the supernatant through three different pore sizes of filter membranes in sequence is that it can prevent large particles of impurities in the feed liquid from quickly clogging the micropores of the 0.22 μm filter membrane, which would result in extremely slow filtration speed or even complete clogging, increasing filter membrane consumption and operating costs. It can also achieve efficient and stable filtration through a step-by-step retention method, maximizing the retention of kelp exovesicles in the filtrate.

[0012] (5) Ultracentrifugation purification: After ultracentrifugation of the filtrate, the supernatant was discarded. The filtrate was centrifuged at a specific ultra-high speed to precipitate the vesicles. The precipitate was resuspended with sterile PBS buffer and ultracentrifuged and washed again under the same conditions to remove co-precipitated soluble proteins and impurities. Finally, purified kelp extracellular vesicle precipitate was obtained. The kelp extracellular vesicle precipitate was resuspended with sterile PBS, aliquoted and stored at -80℃ to obtain kelp extracellular vesicles.

[0013] Furthermore, in step (1), before mixing the kelp with the phosphate buffer, the kelp is first soaked in water and then thoroughly washed until the surface is no longer sticky.

[0014] Furthermore, in step (1), the mass-to-volume ratio of kelp to phosphate buffer is 1:8 to 1:35.

[0015] Furthermore, in step (2), the amount of alginate lyase, cellulase and pectinase added accounts for 0.1%~1.5%, 0.05%~1.5% and 0.01%~1% of the mass of kelp, respectively. First, the addition of these three enzymes can specifically degrade the core components of the kelp cell wall and release extracellular vesicles. The cell wall of kelp is mainly composed of alginate (20%–40% dry weight), cellulose (5%–10% dry weight), and pectin (3%–8% dry weight), forming a dense network structure. This network is the main physical barrier preventing the release of extracellular vesicles. The three enzymes have specific functions: alginate lyase specifically cleaves the β-1,4-glycosidic bonds in alginate molecules, disrupting the rigid framework of the cell wall; cellulase enzymatically breaks down the β-1,4-glycosidic bonds in cellulose, disintegrating the supporting structure of the polysaccharide network; and pectinase enzymatically breaks down the α-1,4-glycosidic bonds in pectin within the cell wall, disrupting the cross-links between polysaccharide chains and further loosening the cell wall structure. The synergistic effect of these three enzymes achieves deep cell wall lysis, causing cell rupture or widening of intercellular spaces, thereby efficiently releasing the extracellular vesicles encapsulated within. Furthermore, enzymatic hydrolysis, as a gentler method of cell wall disruption, effectively protects the structure and activity of the extracellular vesicles. Compared to physical disruption methods (such as high-pressure homogenization and ultrasonic disruption), enzymatic hydrolysis offers gentler reaction conditions, avoiding the damage to the extracellular vesicle membrane structure caused by harsh physical forces. This maximizes the preservation of the integrity of extracellular vesicles and their internal active substances, ensuring the reliability of subsequent detection and applications. Secondly, the action of these three enzymes reduces the viscosity of the homogenate, improving subsequent separation efficiency. After kelp homogenization, free polysaccharides dissolve in the aqueous solution, forming a high-viscosity system. This leads to low particle sedimentation efficiency during differential centrifugation and easy adsorption and retention of extracellular vesicles by the polysaccharide network. After the three enzymes degrade the polysaccharides, large polysaccharides are broken down into smaller oligosaccharides or monosaccharides, significantly reducing the system viscosity. This not only accelerates the separation of the supernatant and precipitate during differential centrifugation but also reduces adsorption loss of extracellular vesicles in the polysaccharide matrix, improving the recovery rate of the target product. Finally, the three enzymes can fully enzymatically hydrolyze large polysaccharide impurities, optimizing subsequent filtration and purification effects. Large polysaccharides in the homogenate can easily clog the filter membrane pores during subsequent gradient filtration, prolonging filtration time. Enzymatic hydrolysis can convert the main macromolecular polysaccharides in the homogenate into smaller molecules. These smaller impurities are easily removed in subsequent separation steps, improving filtration throughput and the purity of the target product.

[0016] Further, the differential centrifugation conditions in step (3) are as follows: at 4℃, centrifuge at 2000×g for 15 min and collect the supernatant; centrifuge at 5000×g for 30 min and collect the supernatant; centrifuge at 10,000×g for 60 min and collect the supernatant. Its core advantage is to remove impurities of different particle sizes step by step, efficiently enrich extracellular vesicles and protect their structural integrity to the greatest extent. First, removing impurities step by step can avoid extracellular vesicles being trapped by co-precipitation. The kelp enzymatic hydrolysis supernatant contains a variety of impurities, whose sedimentation coefficients and particle sizes are significantly different. Gradient centrifugation can achieve "from coarse to fine" directional removal: "centrifugation at 2000×g for 15 min" can remove large particle impurities, including incompletely enzymatically hydrolyzed cell wall fragments, cell clumps, and some organelle residues. These impurities have large particle sizes and fast sedimentation speeds, and can be effectively sedimented at low speeds; if they are not removed first, they will form a dense precipitate layer during subsequent high-speed centrifugation, adsorbing and encapsulating extracellular vesicles, leading to a decrease in recovery rate. Centrifugation at 5000×g for 30 min removes impurities such as polymerized polysaccharide colloids and fragmented subcellular structures (e.g., chloroplast fragments, mitochondrial fragments). These impurities have particle sizes and sedimentation rates between large particles and extracellular vesicles; moderate centrifugation speeds allow for precise retention and further purification of the supernatant. Centrifugation at 10000×g for 60 min removes fine impurity particles, including oligosaccharide aggregates produced by enzymatic hydrolysis, residual enzyme-protein complexes, and small protein aggregates. Extending the centrifugation time ensures sufficient sedimentation of fine impurities while avoiding premature precipitation of extracellular vesicles due to over-centrifugation (100000×g). Furthermore, a 4°C setting protects the structure and activity of extracellular vesicles. The membrane structure of extracellular vesicles consists of a lipid bilayer, which is prone to fusion and rupture at room temperature, leading to leakage of vesicle contents. The low temperature of 4°C reduces the fluidity of membrane lipids, maintaining the morphological integrity of the vesicles. Low temperatures can inhibit the activity of residual proteases and glycosidases in the enzymatic hydrolysis system, preventing these enzymes from degrading active molecules such as proteins and polysaccharides on the vesicle surface, thus ensuring the reliability of subsequent detection and applications. Furthermore, gradient centrifugation parameters are adapted to the sedimentation characteristics of extracellular vesicles. The particle size of exosome-like nanovesicles derived from kelp is typically 30-150 nm. The highest rotational speed in this centrifugation step is 10000×g, which does not reach the sedimentation threshold of the vesicles, achieving the goal of "impurity sedimentation and vesicle suspension," providing a high-purity feed solution for subsequent ultracentrifugation enrichment of vesicles.

[0017] Furthermore, the conditions for ultracentrifugation in step (5) are as follows: centrifuge at 100,000×g for 60 min at 4℃, resuspend the precipitate in phosphate buffer, centrifuge again at 100,000×g for 60 min, and collect the precipitate. The synergistic advantage of the two-step ultracentrifugation is that it takes into account both the recovery rate and purity of extracellular vesicles. Compared with single ultracentrifugation, the two-step centrifugation strategy achieves two-step separation of "enrichment-purification". First, "centrifugation at 100,000×g for 60 min" can target the sedimentation and enrich extracellular vesicles. The particle size of extracellular vesicles derived from kelp is mostly 30-150 nm, and the centrifugal force threshold corresponding to their sedimentation coefficient is about 80,000-120,000×g. Centrifugation at 100,000×g precisely overcomes the buoyancy resistance of vesicles, allowing them to settle fully in the supernatant and form a precipitate. Small molecule impurities (such as oligosaccharides and free proteins) not completely removed in the first centrifugation stage will remain in the supernatant and be removed due to their low sedimentation coefficient. Centrifugation for 60 min ensures that most vesicles in the solution settle completely, reducing the loss of target products due to insufficient centrifugation time, while also avoiding mechanical stress from prolonged centrifugation that could damage the vesicle structure. Secondly, resuspending and then centrifuging again at 100,000×g for 60 min purifies and washes the vesicles, improving their purity. This step is a crucial "washing" step in differential centrifugation for purifying extracellular vesicles, often referred to in the industry as "washing centrifugation." Its function is to: ① remove co-precipitated soluble impurities. The precipitate obtained from the first 100,000×g centrifugation, in addition to the target vesicles, will also adsorb a small amount of co-precipitated soluble impurities such as proteins, polysaccharides, and salt ions. Resuspending the precipitate with phosphate buffer allows these impurities to redissolve in the solution. ② Short-duration high-speed centrifugation achieves "purity upgrade": Centrifuge again at 100,000×g for 60 min. Utilizing the difference in sedimentation rates between vesicles and soluble impurities, the denser vesicles settle rapidly, while the dissolved impurities remain in the supernatant and are discarded.

[0018] A kelp extracellular vesicle prepared by the above method. The kelp-derived extracellular vesicle is isolated from kelp and is a nanoscale vesicle with a typical saucer-like structure. The kelp-derived extracellular vesicle has a small particle size, uniform size, and intact morphology.

[0019] Furthermore, the average particle size of the kelp extracellular vesicles is 100~200 nm, preferably 131.6±3.5 nm.

[0020] Furthermore, the particle number concentration of the kelp-derived extracellular vesicles is 1.1 × 10⁻⁶. 10 particles / mL, protein concentration 0.21 mg / mL. Preferably, the ratio of granules to protein in kelp-derived extracellular vesicles is 5.5 × 10⁻⁶ mg / mL.10 particles / mg.

[0021] An application of the above-mentioned kelp extracellular vesicles as a serum substitute.

[0022] Furthermore, the kelp extracellular vesicles are used in the preparation of culture additives or low-serum culture media for promoting the proliferation of large yellow croaker muscle stem cells and / or maintaining stem cell characteristics.

[0023] Furthermore, kelp extracellular vesicles were added to a low-serum culture medium with a kelp extracellular vesicle concentration of 5 μg / mL and a serum concentration of 5% FBS.

[0024] The beneficial effects of this invention are as follows:

[0025] (1) This invention discloses for the first time extracellular vesicles derived from kelp. These vesicles are nanoscale membrane vesicles with a typical saucer-like morphology and an average particle size of less than 150 nm, which can be effectively taken up by large yellow croaker muscle stem cells.

[0026] (2) The method for isolating extracellular vesicles of kelp provided by this invention is simple, scalable, and uses inexpensive raw materials. Using it in low serum culture medium can significantly reduce dependence on expensive fetal bovine serum, providing a practical solution for cost reduction and efficiency improvement in cell-cultured meat.

[0027] (3) This invention is the first to develop the extracellular vesicles of kelp, a seaweed that is both food and medicine, into an active supplement for the culture of muscle stem cells of aquatic animals (large yellow croaker). It has confirmed that the extracellular vesicles of kelp have multiple biological functions such as promoting the proliferation, migration and maintaining the stemness of muscle stem cells of large yellow croaker, and expanding the application scope of food-derived vesicles in cell agriculture.

[0028] (4) No chemical reagents need to be added during the preparation process of this invention, and the preparation method is simple. Kelp, as a traditional edible and medicinal seaweed, has high safety. Its extracellular vesicles have good biocompatibility and are renewable in origin, which meets the core requirements of the cell culture meat industry for safe, controllable and sustainable culture medium components.

[0029] (5) This invention provides a paradigm for the development of other food-derived vesicles, and the separation method of this invention has universal reference value for the extraction of other polysaccharide-rich plant-derived extracellular vesicles. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The figure shows the analysis results of the extracellular vesicle particle size obtained before and after enzymatic hydrolysis of kelp.

[0032] Figure 2 The figure shows the analysis results of the concentration of extracellular vesicle particles obtained before and after enzymatic hydrolysis of kelp.

[0033] Figure 3 This figure shows the analysis results of the relative purity of extracellular vesicles obtained before and after enzymatic hydrolysis of kelp. ** indicates p < 0.01, meaning there is a highly statistically significant difference between the two groups.

[0034] Figure 4 Transmission electron microscopy images of the extracellular vesicles obtained before and after enzymatic hydrolysis of kelp.

[0035] Figure 5 Laser confocal imaging of the efficient uptake of extracellular vesicles of kelp by muscle stem cells of large yellow croaker.

[0036] Figure 6 The following are statistical graphs showing the effects of kelp extracellular vesicles on the proliferation of large yellow croaker muscle stem cells. Figure a shows the statistical results of the effects of kelp extracellular vesicles on the activity of large yellow croaker muscle stem cells measured by the CCK-8 assay. * indicates p < 0.05, meaning there is a statistically significant difference between the two groups; ** indicates p < 0.01, meaning there is an extremely statistically significant difference between the two groups; *** indicates p < 0.001, but this symbol does not appear in the attached figures. Figure b shows representative fluorescence images of the effect of kelp extracellular vesicles on the proliferation of large yellow croaker muscle stem cells measured by the EdU assay. Figure c shows the quantitative results of the proportion of positive proliferating cells based on the above EdU fluorescence images. * indicates p < 0.05, meaning there is a statistically significant difference between the two groups; ** indicates p < 0.01, meaning there is an extremely statistically significant difference between the two groups.

[0037] Figure 7 Figure 1 shows the effect of kelp extracellular vesicles on the migration ability of large yellow croaker muscle stem cells. Figure 2a shows a representative microscopic image of the effect of kelp extracellular vesicles on the migration ability of large yellow croaker muscle stem cells measured by the scratch assay. Figure 3b shows a statistical graph of the results of quantitative scratch area quantification of the representative scratch assay images. Figure 4c shows a representative microscopic image of the effect of kelp extracellular vesicles on the migration ability of large yellow croaker muscle stem cells measured by the Transwell chamber permeation assay. Figure 5d shows a statistical graph of the results of quantitative transwell permeation cell count. * indicates p < 0.05, that is, there is a statistically significant difference between the two groups, and ** indicates p < 0.01, that is, there is an extremely significant difference between the two groups.

[0038] Figure 8Figure 1 shows the results of the effect of kelp extracellular vesicles on the maintenance of stemness of large yellow croaker muscle stem cells. Figure 2a shows a representative microscopic image of the effect of kelp extracellular vesicles on Pax7 protein expression in large yellow croaker muscle stem cells, investigated by immunostaining. Figure 3b shows the statistical results of the relative fluorescence intensity quantification of the representative images of the above-mentioned Pax7 protein immunostaining. Figure 4c shows the statistical results of the effect of kelp extracellular vesicles on Pax7 gene expression in large yellow croaker muscle stem cells, investigated by RT-qPCR. Figure 5d shows a representative microscopic image of the effect of kelp extracellular vesicles on Myod protein expression in large yellow croaker muscle stem cells, investigated by immunostaining. Figure 6e shows the statistical results of the relative fluorescence intensity quantification of the representative images of the above-mentioned Myod protein immunostaining. Figure 7f shows the statistical results of the effect of kelp extracellular vesicles on Myod gene expression in large yellow croaker muscle stem cells, investigated by RT-qPCR. * indicates p < 0.05, that is, there is a statistically significant difference between the two groups, and ** indicates p < 0.01, that is, there is an extremely significant difference between the two groups.

[0039] In all the above figures, ns indicates no significant difference, * indicates p < 0.05, and ** indicates p < 0.01. Detailed Implementation

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0044] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available or can be prepared by existing methods.

[0046] Example 1: A method for preparing extracellular vesicles of kelp, comprising the following steps:

[0047] (1) Raw material pretreatment: Soak dried kelp in water for 6 hours. After soaking, wash away the dust particles and other impurities on the surface until there is no stickiness on the surface. Add phosphate buffer solution at a ratio of 1:20 and mix well to fully extract the kelp juice and form a suspension.

[0048] (2) Enzymatic hydrolysis: Add 0.3% alginate lyase, 0.2% cellulase and 0.1% pectinase based on the weight of kelp to the homogenate and perform mild enzymatic hydrolysis for 12 h at 37 °C and pH 6.9.

[0049] (3) Primary separation: Transfer the fully digested enzyme solution to a centrifuge tube and perform differential centrifugation. The steps of differential centrifugation are as follows: under 4℃ conditions, centrifuge at 2000×g for 15 min and collect the supernatant for the first time; centrifuge at 5000×g for 30 min and collect the supernatant for the second time; centrifuge at 10,000×g for 60 min and collect the supernatant for the third time.

[0050] (4) Step-by-step filtration: The supernatant after differential centrifugation is filtered sequentially through filter membranes with pore sizes of 0.8 μm, 0.45 μm and 0.22 μm, and the filtrate is collected.

[0051] (5) Ultracentrifugation purification: Transfer the filtrate to an ultracentrifuge tube and centrifuge at 100,000×g for 60 min at 4°C. Resuspend the precipitate obtained by centrifugation with phosphate buffer, filter through a 0.22 μm filter membrane, and centrifuge again at 100,000×g for 60 min. Resuspend the precipitate with sterile PBS and collect it to obtain kelp extracellular vesicles.

[0052] Example 2: This example demonstrates the characterization of the extracellular vesicles of kelp prepared in Example 1.

[0053] 1. Protein concentration of extracellular vesicles obtained before and after enzymatic hydrolysis of kelp.

[0054] The protein concentration of extracellular vesicles obtained before and after enzymatic hydrolysis of the kelp prepared in Example 1 was detected and measured using a BCA kit.

[0055] The results showed that the protein concentration of the kelp extracellular vesicles obtained by enzymatic hydrolysis was 0.21 mg / mL, while the protein concentration of the kelp extracellular vesicles without enzymatic hydrolysis was 0.26 mg / mL, indicating that the extracellular vesicles without enzymatic hydrolysis may contain more extraneous proteins.

[0056] 2. The particle size of extracellular vesicles obtained before and after enzymatic hydrolysis of kelp.

[0057] The particle size of the extracellular vesicles obtained before and after enzymatic hydrolysis of kelp prepared in Example 1 was analyzed and detected by dynamic light scattering. Specifically, the protein concentration of kelp extracellular vesicles was diluted to an appropriate concentration using phosphate buffer, and then added to a particle size distribution dish for detection by dynamic light scattering.

[0058] The results are as follows Figure 1 As shown, the hydrated particle sizes of the extracellular vesicles obtained before and after enzymatic hydrolysis of kelp were 174.30±4.67 nm and 131.60±3.48 nm, respectively. Furthermore, the extracellular vesicles obtained after enzymatic hydrolysis had a relatively low polydispersity index (PDI, 0.136 ±0.016), indicating that the enzymatic hydrolysis method resulted in higher size uniformity of the vesicles.

[0059] 3. The particle concentration of extracellular vesicles obtained before and after enzymatic hydrolysis of kelp.

[0060] The particle concentration of extracellular vesicles obtained before and after enzymatic hydrolysis of kelp prepared in Example 1 was detected by a nanoparticle tracking analyzer as follows: The protein concentration of kelp extracellular vesicles was diluted to an appropriate concentration using ultrapure water and injected into the nanoparticle tracking analyzer for detection.

[0061] The results are as follows Figure 2 As shown, the particle concentrations of extracellular vesicles obtained before and after enzymatic hydrolysis of kelp were 4.9 × 10⁻⁶. 9 particles / mL and 1.1 × 10 10 particles / mL means that the extracellular vesicles obtained after enzymatic hydrolysis have a relatively higher particle concentration.

[0062] 4. Purity of extracellular vesicles obtained before and after enzymatic hydrolysis of kelp.

[0063] The purity of the extracellular vesicles obtained before and after enzymatic hydrolysis of kelp prepared in Example 1 was calculated according to the following formula: Purity = Kelp extracellular vesicle particle concentration (particles / mL) / Kelp extracellular vesicle protein content (mg / mL).

[0064] The results are as follows Figure 3 As shown, the ratio of particle concentration to protein concentration of extracellular vesicles obtained after enzymatic hydrolysis was significantly higher than that of vesicles before enzymatic hydrolysis, indicating that enzymatic hydrolysis of kelp can improve the purity and yield of extracellular vesicles.

[0065] 5. Microscopic morphology of extracellular vesicles obtained before and after enzymatic hydrolysis of kelp.

[0066] The microscopic morphology of the extracellular vesicles obtained before and after enzymatic hydrolysis of kelp prepared in Example 1 was examined by transmission electron microscopy. The specific operation steps are as follows: 1. Adsorption: 10 μL of kelp extracellular vesicle suspension was pipetted onto a pre-prepared Parafilm sealing film. The copper mesh was placed face down, allowing it to naturally adsorb the suspension droplets for 10 minutes. Then, excess droplets were removed with filter paper strips, and the membrane was dried under a lamp. 2. Staining: 10 μL of 2% phosphotungstic acid solution was pipetted onto the sealing film. The copper mesh, after adsorption, was placed face down with the staining solution and inverted to stand for 5 minutes. 3. Drying: Excess droplets were removed with filter paper strips, and the membrane was air-dried under an incandescent lamp. 4. Observation and photography: Photos were taken under a transmission electron microscope.

[0067] The results are as follows Figure 4 As shown, typical saucer-shaped extracellular vesicles can be extracted from kelp before and after enzymatic hydrolysis. However, the nanovesicles extracted by enzymatic hydrolysis have higher purity, more typical membrane structure, uniform dispersion, and complete morphology, indicating that the extracellular vesicles obtained by enzymatic hydrolysis of kelp have significant advantages. Therefore, Example 1 was selected as the preferred method for extracting extracellular vesicles from kelp and subsequent analysis was conducted.

[0068] Example 3: In this example, the effects of kelp extracellular vesicles prepared in Example 1 on the proliferation, migration and stemness maintenance of large yellow croaker muscle stem cells were investigated using in vitro cell experiments.

[0069] 1. Laser confocal imaging of muscle stem cells in large yellow croaker taking up extracellular vesicles of kelp.

[0070] Fluorescent images of the kelp extracellular vesicles prepared in Example 1 being taken up by large yellow croaker muscle stem cells were detected using laser confocal microscopy. The specific steps were as follows: Kelp extracellular vesicles were incubated with PKH67 fluorescent dye at 37°C for 30 minutes. Residual dye was then separated by centrifugation at 100,000 × g for 1 hour to obtain PKH67-labeled kelp extracellular vesicles. These vesicles were then co-incubated with large yellow croaker muscle stem cells for 12 hours. Cells were then fixed with 4% paraformaldehyde for 15 minutes, and subjected to 0.1% trition X-100 for 10 minutes to lyse the cell membrane. The cytoskeleton was stained with rhodamine-labeled phalloidin, and the cell nuclei were stained with DAPI dye. Finally, cell uptake was observed under a confocal laser scanning microscope.

[0071] The results are as follows Figure 5 As shown, PKH67 green fluorescently labeled kelp extracellular vesicles were localized in the cytoplasm of large yellow croaker muscle stem cells, indicating that large yellow croaker muscle stem cells can efficiently take up kelp extracellular vesicles.

[0072] 2. Effects of kelp extracellular vesicles on the proliferation capacity of large yellow croaker muscle stem cells.

[0073] The effects of extracellular vesicles from kelp on the proliferation of muscle stem cells in large yellow croaker were detected using the CCK-8 and EdU assays, respectively. The specific experimental steps are as follows:

[0074] CCK-8 assay: Large yellow croaker muscle stem cells were seeded at a density of 10,000 cells / well in 96-well plates and cultured in DMEM / F12 medium containing 5% FBS at 28°C for 24 h. The culture medium was then replaced with medium containing different concentrations of kelp extracellular vesicles (0 μg / mL, 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL), with three replicates for each treatment. After culturing for 24, 48, and 72 hours respectively, the viability of large yellow croaker muscle stem cells was detected using a CCK-8 assay kit (CCK-8assay, beyotime, C0042) to observe the effect of kelp extracellular vesicles on the proliferation capacity of large yellow croaker muscle stem cells.

[0075] The results are as follows Figure 6 As shown in Figure a, after adding extracellular vesicles of kelp to 5% FBS medium, cell viability continuously increased with increasing culture time, indicating that the muscle cells of large yellow croaker slowly adapted to the low serum environment containing TLEVs and began to proliferate rapidly after complete adaptation. Among them, the 5 μg / mL kelp extracellular vesicle dose group can obtain the greatest proliferation effect at a smaller dose.

[0076] EdU method: Large yellow croaker muscle stem cells were seeded at a density of 50,000 cells / well in confocal microplates and cultured in DMEM / F12 medium containing 5% FBS at 28°C for 24 h. The culture medium was then replaced with medium containing 5 μg / mL kelp extracellular vesicles. Each treatment was performed in 6 replicates. After culturing for another 24 hours, the newly proliferating large yellow croaker muscle stem cells were detected using the EdU kit (Click™ EdU-488assay, beyotime, C0071S) to observe the effect of kelp extracellular vesicles on the proliferation capacity of large yellow croaker muscle stem cells.

[0077] The results are as follows Figure 6As shown in Figures b and c, compared with the 5% FBS group, the proportion of EdU-positive proliferating cells in the 5% FBS group containing 5 μg / mL kelp extracellular vesicles was significantly increased, indicating that 5 μg / mL has a significant promoting effect on the proliferation capacity of large yellow croaker muscle stem cells.

[0078] 3. Effects of kelp extracellular vesicles on the migration ability of large yellow croaker muscle stem cells.

[0079] The effects of kelp extracellular vesicles on the migration ability of large yellow croaker muscle stem cells were detected using the scratch assay and Transwell assay, respectively. The specific experimental steps are as follows:

[0080] Scratch assay: Large yellow croaker muscle stem cells were subjected to a 5×10⁻⁶ scratch assay. 5 Cells were seeded at a density of 1 cell per well in 6-well plates and cultured for 24 hours until 100% confluence was achieved. Mitomycin C (1 μg / mL) was added for 30 minutes to eliminate the influence of cell proliferation. Cells were then scratched with a 200 μL pipette tip, and the original medium was replaced with 3% FBS containing 3% FBS and 5 μg / mL kelp extracellular vesicles. Cells were cultured for 24 and 48 hours, and wound healing was observed to assess the effect of kelp extracellular vesicles on the migration ability of large yellow croaker muscle stem cells.

[0081] The results are as follows Figure 7 As shown in Figures a and b, representative images of scratch wound healing show that cells in both groups migrated significantly over time. The experimental group, which received 5 μg / mL of kelp extracellular vesicles, healed the entire scratch wound faster than the control group. Figure 7 (Figure a) Furthermore, quantitative analysis of the cell scratch area using ImageJ software showed that the cell migration area in the TLEVs-treated group was significantly higher than that in the control group (Figure a). Figure 7 Figure b shows that 5 μg / mL of kelp extracellular vesicles can promote the migration ability of large yellow croaker muscle stem cells.

[0082] Transwell assay: In the experiment, large yellow croaker muscle stem cells were used at a rate of 5 × 10⁻⁶. 4 Cells were seeded at a density of 1 cell per well in the upper layer of a 24-well cell culture plate with a breathable membrane, and then culture medium containing 5 μg / mL kelp extracellular vesicles was placed in the lower layer of the cell culture plate. After 24 hours of culture, the permeabilized cells were stained and counted to assess the effect of kelp extracellular vesicles on the migration ability of large yellow croaker muscle stem cells.

[0083] The results are as follows Figure 7 As shown in Figures c and d, the number of cells crossing the basement membrane in the control group and the TLEVs group were 72.2 and 82.2 cells / field, respectively. Figure 7Figures c and d show that 5 μg / mL of kelp extracellular vesicles can enhance the migration ability of large yellow croaker muscle stem cells.

[0084] 4. Effects of kelp extracellular vesicles on the maintenance of stemness of muscle stem cells in large yellow croaker.

[0085] The effects of kelp extracellular vesicles on stem cell gene expression in large yellow croaker muscle stem cells were detected by immunofluorescence staining and RT-qPCR, respectively. The specific experimental steps are as follows: Cell culture: Large yellow croaker muscle stem cells were cultured at 5×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 10 cells / well in 24-well plates and cultured for 24 h. The culture medium was then replaced with medium containing 5% FBS and 5 μg / mL kelp extracellular vesicles in 5% FBS. Cells were cultured continuously for 3 generations. The expression of stemness marker genes was then quantified by immunofluorescence staining or RT-qPCR. Immunostaining: Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% skim milk powder for 2 h. Subsequently, cells were incubated overnight at 4°C with Pax7 and / or Myod1 antibodies, and then incubated at room temperature with m-IgGκBP-PE for 2 h. Finally, the nuclei were stained using DAPI staining. RT-qPCR: RNA was extracted from large yellow croaker muscle stem cells using a total RNA extraction kit (Total RNA Extractor, Sangon Biotech, B511311), and the RNA was reverse transcribed into cDNA using a cDNA synthesis kit (BeyoRT™ Q cDNA, beyotime, D7190M). RT-qPCR was then performed using a StepOnePlus™ real-time quantitative PCR system with SYBR Green qPCR Mix premix (BeyoFast™ SYBR Green qPCR Mix, beyotime, D7260).

[0086] The results are as follows Figure 8 As shown in Figures a and d, the nuclear protein staining of Pax7 and MyoD, marker proteins of large yellow croaker muscle stem cells, co-localizes with the cell nucleus. Quantitative analysis of their fluorescence intensity shows that after three consecutive generations of culture under low serum conditions, the fluorescence intensity of Pax7 in large yellow croaker muscle stem cells shows a decreasing trend, but there is no significant difference. Figure 8 (Figure b) shows that the expression level of MyoD was significantly suppressed. Figure 8(See Figure e). Adding 5 μg / mL of kelp extracellular vesicles to the medium had no effect on Pax7, but significantly increased the MyoD expression level in cells cultured in 5% FBS medium, indicating that adding 5 μg / mL of kelp extracellular vesicles can maintain the stability of gene expression in large yellow croaker cells. RT-PCR results showed similar conclusions; adding 5 μg / mL of kelp extracellular vesicles to low serum medium significantly upregulated the expression levels of both Pax7 and MyoD genes in large yellow croaker muscle stem cells. Figure 8 (See Figures c and f). Therefore, 5 μg / mL of kelp extracellular vesicles can maintain stemness during long-term cell culture.

[0087] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0088] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0089] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. An application of kelp extracellular vesicles as a serum substitute, characterized in that: This material is used to promote the proliferation of large yellow croaker muscle stem cells and maintain their stem cell characteristics; its preparation method includes the following steps: (1) Raw material pretreatment: Wash and crush the dried kelp, mix it with phosphate buffer solution, extract the kelp juice, and form a suspension; (2) Enzymatic hydrolysis: Add a complex enzyme solution of alginate lyase, cellulase and pectinase to the above suspension, and carry out a mild enzymatic hydrolysis reaction under the conditions of 25-45℃, pH 6.0-8.0 and hydrolysis time of 6-12 h. (3) Primary separation: After the enzymatic hydrolysis reaction is completed, the supernatant is collected after differential centrifugation; (4) Stepwise filtration: The supernatant above was filtered sequentially through filter membranes with pore sizes of 0.8 μm, 0.45 μm and 0.22 μm, and the filtrate was collected; (5) Ultracentrifugation purification: After ultracentrifugation of the filtrate, the supernatant was discarded; the precipitate was resuspended with sterile PBS buffer, and ultracentrifuged and washed again under the same conditions to finally obtain purified kelp extracellular vesicle precipitate. The kelp extracellular vesicle precipitate was resuspended with sterile PBS, aliquoted and stored at -80℃ to obtain kelp extracellular vesicles.

2. The application as described in claim 1, characterized in that: Before mixing the kelp with the phosphate buffer in step (1), soak the kelp in water until it expands, and then wash it thoroughly until there is no stickiness on the surface.

3. The application as described in claim 1, characterized in that: In step (1), the mass-to-volume ratio of kelp to phosphate buffer is 1:8 to 1:

35.

4. The application as described in claim 1, characterized in that: Step (2) The amount of alginate lyase, cellulase and pectinase added accounts for 0.1%~1.5%, 0.05%~1.5% and 0.01%~1% of the mass of kelp, respectively.

5. The application as described in claim 1, characterized in that: The differential centrifugation conditions in step (3) are as follows: at 4℃, centrifuge at 2000×g for 15min and collect the supernatant; centrifuge at 5000×g for 30min and collect the supernatant; centrifuge at 10,000×g for 60min and collect the supernatant.

6. The application as described in claim 1, characterized in that: Step (5) The conditions for ultracentrifugation are as follows: centrifuge at 100,000×g for 60 min at 4℃, resuspend the precipitate with phosphate buffer, centrifuge again at 100,000×g for 60 min, and collect the precipitate.

7. The application as described in claim 1, characterized in that: Laminaria extracellular vesicles were added to a low-serum culture medium with a lamina extracellular vesicle concentration of 5 μg / mL and a serum concentration of 5% FBS.

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