Dendrobium nobile stem cell exosome-producing culture medium and application thereof

Abstract

The application provides a culture medium for producing dendrobium stem cell exovesicles and an application thereof, relates to the technical field of biology, and the culture medium for producing dendrobium stem cell exovesicles comprises 0.1-0.5 mg / L naphthalene acetic acid, 0.05-0.2 mg / L thidiazuron, 0.5-5 muM sodium selenate and 1-10 mg / L glutathione. The culture medium for producing dendrobium stem cell exovesicles and the method for preparing dendrobium stem cell exovesicles by using the culture medium for producing dendrobium stem cell exovesicles provided by the application have made significant progress in the yield and quality of dendrobium stem cell exovesicles. Meanwhile, the prepared dendrobium stem cell exovesicles have significant DPPH free radical scavenging capacity and improve the healing capacity of skin fibroblasts.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a culture medium for producing Dendrobium stem cell vesicles and its application. Background Technology

[0002] Dendrobium is a traditional and precious Chinese medicinal herb, and its stem cells contain abundant medicinal active ingredients. Extracellular vesicles (EVs) are nanoscale vesicles actively secreted by cells, carrying important biological information molecules such as proteins, nucleic acids, and lipids, playing a crucial role in intercellular communication. Recent studies have found that EVs derived from Dendrobium stem cells possess a variety of remarkable biological functions, including promoting the proliferation and migration of skin fibroblasts, accelerating wound healing, and anti-inflammatory effects, showing great potential in the development of high-end cosmetics, wound repair, and immunomodulatory drugs.

[0003] Currently, methods for obtaining plant extracellular vesicles (EVs) are mainly limited: 1) Direct extraction from fresh Dendrobium tissues, resulting in extremely low yields, limited by season and production location, and unable to be scaled up; 2) Solid-state culture, which is complex, prone to contamination, and difficult to control; 3) Using traditional plant cell suspension cultures (such as MS and B5 media), but these media are not designed for extracellular vesicle production and generally suffer from low EV yields and unclear active ingredients. More importantly, many culture systems rely on fetal bovine serum (FBS), and the exogenous EVs contained in the serum can seriously interfere with the purification and identification of target EVs, and bring potential risks to pathogen safety and immunogenicity.

[0004] Therefore, there is an urgent need in this field to develop a serum-free, clearly defined culture medium MSH that can simultaneously and efficiently promote the growth of Dendrobium stem cells and induce them to secrete large amounts of highly active EVs, in order to overcome the technical bottleneck of industrial production of Dendrobium stem cell EVs. Summary of the Invention

[0005] This invention provides a culture medium for producing Dendrobium stem cell extravesicles and its application, which solves the defects of the prior art, such as low yield and purity of Dendrobium stem cell extravesicles, complex and easily contaminated culture process, and unclear active ingredients. It provides a serum-free, clearly defined culture medium MSH that can simultaneously and efficiently promote the growth of Dendrobium stem cells and induce them to secrete a large number of highly active extracellular vesicles, as well as a matching culture method.

[0006] In a first aspect, the present invention provides a culture medium for producing Dendrobium stem cell vesicles, characterized in that it comprises: 0.1-0.5 mg / L naphthaleneacetic acid (NAA), 0.05-0.2 mg / L thidiazuron (TDZ), 0.5-5 μM sodium selenate and 1-10 mg / L glutathione.

[0007] In the culture medium for producing Dendrobium stem cell vesicles provided by this invention, the concentration of naphthaleneacetic acid is 0.1-0.5 mg / L, for example, it can be 0.1 mg / L, 0.15 mg / L, 0.2 mg / L, 0.25 mg / L, 0.3 mg / L, 0.35 mg / L, 0.4 mg / L, 0.45 mg / L, or 0.5 mg / L, but is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0008] In the culture medium for producing Dendrobium stem cell vesicles provided by this invention, the concentration of thidiazuron is 0.05-0.2 mg / L, for example, it can be 0.05 mg / L, 0.08 mg / L, 0.10 mg / L, 0.13 mg / L, 0.15 mg / L, 0.17 mg / L, or 0.20 mg / L, but is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0009] In the culture medium for producing Dendrobium stem cell vesicles provided by this invention, the concentration of sodium selenate is 0.5-5 μM, for example, it can be 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, or 5.0 μM, but is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0010] In the culture medium for producing Dendrobium stem cell vesicles provided by this invention, the concentration of glutathione is 1-10 mg / L, for example, it can be 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, but is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0011] The culture medium for producing Dendrobium officinale stem cell extravesicles provided by this invention uses a mixture of naphthaleneacetic acid (NAA) and thidiazuron (TDZ). The preferred concentration range is NAA 0.1-0.5 mg / L and TDZ 0.05-0.2 mg / L. This specific ratio not only synergistically promotes rapid stem cell proliferation but also generates a "mild stress" effect, shifting the cell's metabolic focus from simple growth to the synthesis and secretion of functional extracellular vesicles. Sodium selenate (0.5-5 μM) and glutathione (1-10 mg / L) are added. This combination effectively removes reactive oxygen species (ROS) accumulated during culture, reduces oxidative stress damage to cells, and ensures the integrity of the extracellular vesicle membrane structure and the stability of internal active substances. The culture medium of this invention is completely free of animal serum, fundamentally eliminating exogenous contamination, simplifying downstream purification steps, and ensuring the safety of the final product.

[0012] Preferably, the culture medium for producing Dendrobium stem cell vesicles further includes 1-10 mg / L of Dendrobium polysaccharide hydrolysis product, wherein the Dendrobium polysaccharide hydrolysis product is an oligosaccharide fragment obtained by hydrolyzing Dendrobium polysaccharide with cellulase or pectinase.

[0013] The concentration of Dendrobium polysaccharide hydrolysate in the culture medium for producing Dendrobium stem cell vesicles provided in this protocol is 1-10 mg / L, for example, it can be 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, but is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0014] The culture medium for producing Dendrobium stem cell extravesicles in this scheme also includes 1-10 mg / L of Dendrobium polysaccharide hydrolysate. The Dendrobium polysaccharide hydrolysate is an oligosaccharide fragment obtained by hydrolyzing Dendrobium polysaccharide with cellulase or pectinase. The Dendrobium polysaccharide hydrolysate at a concentration of 1-10 mg / L acts as a signaling molecule, which can simulate the damage or stress response of plants under natural conditions and specifically activate the pathways related to intracellular and extracellular vesicle biosynthesis and secretion.

[0015] Preferably, the culture medium for producing Dendrobium stem cell vesicles includes: 0.2-0.5 mg / L naphthaleneacetic acid, 0.1-0.2 mg / L thidiazuron, 2-5 μM sodium selenate, 2-8 mg / L glutathione, and 5-10 mg / L Dendrobium polysaccharide hydrolysate.

[0016] Preferably, the Dendrobium is selected from at least one of Dendrobium officinale, Dendrobium huoshanense, Dendrobium nobile, Dendrobium purpureus, Dendrobium chrysanthum, Dendrobium chrysanthum, and Dendrobium sphaeroides.

[0017] Preferably, the culture medium for producing Dendrobium stem cell vesicles also includes macro-elements, micro-elements, iron sources, organic components, and carbon sources.

[0018] More preferably, the carbon source is sucrose.

[0019] In a second aspect, the present invention provides a method for preparing Dendrobium stem cell vesicles, wherein Dendrobium stem cells are cultured in suspension using the culture medium for producing Dendrobium stem cell vesicles as described in the first aspect above.

[0020] This invention provides a method for preparing Dendrobium stem cell extravesicles. Using the culture medium for producing Dendrobium stem cell extravesicles described in the first aspect above, Dendrobium stem cells are cultured in suspension. This culture medium significantly increases the biomass of Dendrobium stem cells, and the yield of extravesicles is 3-5 times higher than that of traditional MS culture medium. The obtained extravesicles exhibit significantly enhanced biological functions, demonstrating superior effects in promoting collagen synthesis and migration in human skin fibroblasts compared to products from traditional methods. The serum-free system with clearly defined chemical components ensures the purity of the product and its safety for clinical application. The fixed formulation and controllable process result in minimal batch-to-batch variation (<15%), making it highly suitable for large-scale, standardized production. The high yield and efficient purification process significantly reduce unit costs, demonstrating extremely high commercial transformation value.

[0021] Preferably, the method for preparing the Dendrobium stem cell vesicles includes: Logarithmic growth phase Dendrobium stem cells were inoculated at 80-120 g / L (fresh weight) into the culture medium for producing Dendrobium stem cell vesicles as described in any of claims 1-4, and cultured in the dark at 23-28℃ and 100-130 rpm to obtain Dendrobium stem cell vesicles. Preferably, the method for culturing Dendrobium stem cells includes: inoculating Dendrobium callus at 20-40 g / L (fresh weight) into the culture medium containing Dendrobium stem cell-producing vesicles as described in any one of claims 1-4, introducing sterile air at a rate of 5-10 L / min, culturing at a low light level below 500 lux and at 23-27°C for 15-45 days, and obtaining Dendrobium stem cells through 3-5 subcultures.

[0022] Preferably, the dark culture time is 15-45 days.

[0023] Preferably, the relative humidity of the culture environment for dark culture is controlled at 50%-60%.

[0024] As a preferred method, after dark culture, the culture is filtered, the filtrate is centrifuged to remove impurities, and the supernatant is ultracentrifuged at 80,000-100,000×g to collect crude extracellular vesicles. After resuspending, the vesicles are purified by size exclusion chromatography to obtain Dendrobium stem cell extracellular vesicles.

[0025] In some embodiments of the present invention, the method for preparing the Dendrobium stem cell vesicles includes: (1) Preparation of sterile explants: Dendrobium stem segments, pods, protocorms, callus tissue or bottle seedlings are sterilized; (2) Callus induction: Sterile explants were inoculated into solid culture medium and cultured in the dark at 25±2℃ for 15 to 30 days. After 2 to 3 subcultures, loose, yellowish-white, and undifferentiated Dendrobium callus tissue was obtained. (3) Suspension subculture: Inoculate callus tissue at 30 g / L (fresh weight) into the culture medium (liquid culture medium) for producing Dendrobium stem cells as described in the first aspect above, introduce sterile air at 5-10 L / min, culture at low light (below 500 lux) and 25±2℃ for 15-45 days, and obtain Dendrobium stem cells after 3-5 subcultures; (4) Stem cell culture and EV extraction: Dendrobium stem cells in the logarithmic growth phase were inoculated at 100 g / L (fresh weight) into Erlenmeyer flasks containing MSH (liquid culture medium) for producing Dendrobium stem cell vesicles as described in the first aspect above, and cultured in the dark at 25°C and 110 rpm for 30 days; the culture was filtered through a 400-mesh sieve, and the filtrate was centrifuged at 300×g, 2000×g, and 10,000×g to remove impurities. The supernatant was ultracentrifuged at 100,000×g for 70 minutes to collect the crude extracellular vesicles. After resuspending in PBS, the vesicles were purified by size exclusion chromatography to obtain high-purity Dendrobium stem cell EVs. The solid culture medium contains the following basic components: potassium nitrate 900 mg / L, ammonium nitrate 800 mg / L, potassium dihydrogen phosphate 85 mg / L, magnesium sulfate heptahydrate 185 mg / L, anhydrous calcium chloride 220 mg / L, ferrous sulfate 27.8 mg / L, disodium oxalate tetraacetate 37.3 mg / L, manganese sulfate 22.3 mg / L, boric acid 6.2 mg / L, 6-benzylaminopurine (concentration range 0–2.0 mg / L), potassium iodide 8.4 mg / L, sodium molybdate 0.26 mg / L, copper sulfate 0.026 mg / L, cobalt chloride 0.026 mg / L, inositol 100 mg / L, thioammonium hydrochloride 0.1 mg / L, nicotinic acid 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, glycine 0.2 mg / L, naphthaleneacetic acid (concentration range 0–2.0 mg / L), 2,4- Dichlorophenoxyacetic acid (concentration range 0–2.0 mg / L) was used, with the pH of the basic component system being 4.5–6.0. ​​Subsequently, 0–150 g / L mashed potatoes, 0–100 g / L mashed bananas, and 5–30 g / L sucrose were added, and the mixture was thoroughly stirred to ensure all components were homogeneous. The solution was prepared in 30–100 mL volumes per bottle, with 0.5–1 g of agar powder added per bottle. The solution was then sterilized at 120–125°C for 30–35 minutes. The culture medium MSH (per liter) for producing Dendrobium stem cell vesicles is based on macro-elements, micro-elements, iron source, organic components and sucrose, containing 0.1-0.5 mg / L naphthaleneacetic acid (NAA), 0.05-0.2 mg / L thidiazuron (TDZ), 1-10 mg / L Dendrobium polysaccharide enzymatic hydrolysate, 0.5-5 μM sodium selenate and 1-10 mg / L glutathione.

[0026] In some embodiments of the present invention, the method for preparing the Dendrobium stem cell vesicles includes: (1) Preparation of Dendrobium sterile explants: Dendrobium stem segments, Dendrobium pods, sterile Dendrobium protocorms, Dendrobium callus tissue, and Dendrobium seedlings were sterilized; (2) Callus induction by solid culture medium: Dendrobium sterile explants were inoculated into solid culture medium and cultured in a dark room at 25±2℃ for 15 to 30 days. After 2 to 3 subcultures, loose, yellowish-white, undifferentiated Dendrobium callus tissue could be obtained. Suspension subculture: The callus obtained in step (2) is inoculated into the culture medium (liquid culture medium) for producing Dendrobium stem cell vesicles as described in the first aspect above. The inoculation amount is 30 g / L of fresh weight. Sterile air filtered by an air filter is introduced at a flow rate of 5-10 L / min. The culture is carried out at 25±2℃ for 15-45 days under a weak light environment of less than 500 lux. Then, after 3-5 subcultures, Dendrobium single-cell seeds can be obtained. (4) Suspension culture and EV extraction of Dendrobium stem cells: Vigorously growing Dendrobium stem cells in the logarithmic growth phase were inoculated at a rate of 100 g / L (fresh weight) into Erlenmeyer flasks containing the Dendrobium stem cell extracellular vesicles described in the first aspect above (liquid culture medium). The flasks were placed in a shaker at 25°C and 110 rpm for dark culture for 14 days. After culture, the cell clusters were removed by filtration through a 400-mesh sieve, and the filtrate was collected. The filtrate was centrifuged at 300×g for 10 minutes at 4°C to remove dead cells; then centrifuged at 2000×g for 20 minutes to remove cell debris; and finally centrifuged at 10,000×g for 30 minutes to remove large vesicles. The supernatant was ultracentrifuged at 100,000×g for 70 minutes at 4°C, and the precipitate was the crude extracellular vesicle. The precipitate was resuspended in PBS and further purified by size exclusion chromatography (such as the qEV series) to obtain high-purity Dendrobium stem cell EVs.

[0027] The solid culture medium is prepared by mixing the following components: basic components (potassium nitrate 900 mg / L, ammonium nitrate 800 mg / L, potassium dihydrogen phosphate 85 mg / L, magnesium sulfate heptahydrate 185 mg / L, anhydrous calcium chloride 220 mg / L, ferrous sulfate 27.8 mg / L, disodium oxalate tetraacetate 37.3 mg / L, manganese sulfate 22.3 mg / L, boric acid 6.2 mg / L, 6-benzylaminopurine (concentration range 0–2.0 mg / L), potassium iodide 8.4 mg / L, sodium molybdate 0.26 mg / L, copper sulfate 0.026 mg / L, cobalt chloride 0.026 mg / L, inositol 100 mg / L, thioammonium hydrochloride 0.1 mg / L, nicotinic acid 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, glycine 0.2 mg / L, naphthaleneacetic acid (concentration range 0–2.0 mg / L), 2,4- Dichlorophenoxyacetic acid (concentration range 0–2.0 mg / L) was used to adjust the pH of the basic component system to the range of 4.5–6.0. ​​Then, mashed potatoes (0–150 g / L) and mashed bananas (0–100 g / L) were added, along with sucrose (concentration 5–30 g / L). The mixture was thoroughly stirred until all components were homogeneous. The solution was prepared in 30–100 mL increments per bottle, with 0.5–1 g of agar powder added per bottle. The prepared solution was then poured into 300–650 mL breathable glass bottles. The bottles were then subjected to high-temperature, high-pressure steam sterilization at 120–125°C for 30–35 minutes, followed by natural cooling after sterilization.

[0028] The culture medium MSH for producing Dendrobium stem cell vesicles contains the following basic components (per liter): macroelements: potassium nitrate 2500 mg, ammonium dihydrogen phosphate 300 mg, magnesium sulfate 400 mg, calcium chloride 200 mg; microelements: manganese sulfate 10 mg, zinc sulfate 1 mg, boric acid 5 mg, copper sulfate 0.2 mg, sodium molybdate 0.1 mg, cobalt chloride 0.1 mg, potassium iodide 1 mg; iron source: disodium ferric ethylenediaminetetraacetate 20 mg; organic components: inositol 1000 mg, nicotinic acid 5 mg, pyridoxine hydrochloride 0.5 mg, thiamine hydrochloride 5 mg, glycine 2 mg; carbon source: sucrose 30 g. Plant growth regulators: naphthaleneacetic acid (NAA) 0.1-0.5 mg / L and thidiazuron (TDZ) 0.05-0.2 mg / L; inducers: Dendrobium polysaccharide hydrolysate 1-10 mg / L; antioxidants: sodium selenate 0.5-5 μM and glutathione 1-10 mg / L.

[0029] In a third aspect, the present invention provides a Dendrobium stem cell vesicle, which is prepared by the method for preparing Dendrobium stem cell vesicles described in the second aspect above.

[0030] In a fourth aspect, the present invention provides the application of the Dendrobium stem cell vesicles described in the third aspect above in the preparation of cosmetics or pharmaceuticals with skin repair, anti-inflammatory or immunomodulatory functions.

[0031] Compared with the prior art, the beneficial technical effects of the present invention are as follows: The culture medium for producing Dendrobium stem cell vesicles and the method for preparing Dendrobium stem cell vesicles using the culture medium provided by this invention have achieved significant improvements in both the yield and quality of Dendrobium stem cell vesicles. Furthermore, the prepared Dendrobium stem cell vesicles exhibit a significant ability to scavenge DPPH free radicals and enhance the healing ability of skin fibroblasts. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 This is a graph showing the changes in the biomass (dry weight) of Dendrobium stem cells in the experimental and control groups during days 0-30 of Example 2.

[0034] Figure 2 This is a transmission electron microscope (TEM) image of Dendrobium stem cells EVs obtained in the experimental group of Example 2.

[0035] Figure 3 The image shows the particle size (NTA) results of Dendrobium stem cell EVs obtained from the experimental and control groups in Example 2.

[0036] Figure 4 The graph shows the DPPH free radical scavenging ability of Dendrobium stem cells EVs prepared in the experimental and control groups of Example 2.

[0037] Figure 5 The image shows the cell scratch test results of Dendrobium stem cell EVs prepared in the experimental group of Example 2 and the control group in PBS buffer. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0039] Example 1 This embodiment provides a culture medium (MSH) for producing Dendrobium stem cell vesicles. (I) Preparation of experimental materials and instruments Reagents and consumables: Potassium nitrate, ammonium dihydrogen phosphate, magnesium sulfate, calcium chloride, and other macro-element reagents (purity ≥99.0%, Sigma-Aldrich); manganese sulfate, zinc sulfate, boric acid, and other trace element reagents (analytical grade, Sinopharm Group); ferric disodium ethylenediaminetetraacetate (Fe-EDTA, biotechnology grade); organic components such as inositol, nicotinic acid, pyridoxine hydrochloride, thiamine hydrochloride, glycine, etc. (biochemical reagents, Amresco); sucrose (chromatographic grade, Sinopharm Group); naphthaleneacetic acid (NAA), thiazuron (TDZ) (plant growth regulators, purity ≥98%, Sigma-Aldrich); Dendrobium officinale polysaccharide enzymatic hydrolysis products (self-made, obtained by hydrolyzing Dendrobium officinale polysaccharide with cellulase, oligosaccharide fragment molecular weight 300-3000 Da); sodium selenate, glutathione (antioxidant, purity ≥99%, Sigma-Aldrich); double-distilled water (ddH2O, laboratory-made, conductivity ≤10 μS / cm); 1M Potassium hydroxide (KOH) solution (prepared fresh); sterile PBS buffer (standard formulation: 8.00 g sodium chloride, 0.20 g potassium chloride, 1.44 g disodium hydrogen phosphate, and 0.24 g potassium dihydrogen phosphate per liter, pH adjusted to 7.4; sterilized by filtration using a 0.22 μm filter membrane, and dispensed and stored in a laminar flow hood); 0.22 μm sterile filter membrane (polyethersulfone, Millipore); sterile centrifuge tubes, Erlenmeyer flasks, beakers, pipettes, and other consumables (Corning).

[0040] The specific preparation method of Dendrobium polysaccharide enzymatic hydrolysis product is as follows: Dendrobium powder was extracted in hot water at 90℃ for 2.5 hours with a material-to-liquid ratio of 1:25 (g / mL). After concentration, 95% ethanol was added at a ratio of 1:4 (v / v) to precipitate crude polysaccharide. Subsequently, the crude polysaccharide was prepared into a 2.0% (w / v) solution as substrate, and 1.5% (by mass) of cellulase was added. The solution was precisely enzymatically hydrolyzed for 5 hours at pH 5.0 and 50℃, and the enzyme was inactivated in a 90℃ water bath for 10 minutes. Finally, the reaction solution was centrifuged at 8000 rpm for 15 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and freeze-dried to obtain the target product.

[0041] Instruments and equipment: Electronic analytical balance (accuracy 0.001 g, Mettler Toledo); pH meter (accuracy ±0.01, Sartorius); Autoclave (MLS-3750, Panasonic); Clean bench (SW-CJ-2FD, Suzhou Jingantai); Thermostatic shaker (ZWY-2102C, Zhicheng); Ultracentrifuge (Optima L-100K, Beckman); Nanoparticle tracking analyzer (NTA 3.0, Malvern); Transmission electron microscope (TEM, JEM-1230, JEOL); Western blot system (Bio-Rad); Microplate reader (Infinite M200 PRO, Tecan); Biosafety cabinet (BSC-1300IIA2, Suzhou Jingantai).

[0042] (II) Basal culture medium group distribution Precise weighing was performed in a clean bench according to the formula ratio of MSH culture medium for producing Dendrobium stem cell vesicles. First, a clean and dry 1L sterile beaker was selected, placed on an electronic analytical balance and zeroed. The macro-element components were weighed in sequence: potassium nitrate 2500 mg, ammonium dihydrogen phosphate 300 mg, magnesium sulfate 400 mg, and calcium chloride 200 mg. After adding each reagent, the beaker was gently shaken to avoid reagent accumulation.

[0043] Then, the trace element components were added: 10 mg manganese sulfate, 1 mg zinc sulfate, 5 mg boric acid, 0.2 mg copper sulfate, 0.1 mg sodium molybdate, 0.1 mg cobalt chloride, and 1 mg potassium iodide. Since some trace elements were added in very small amounts, a pipette was used to assist in their addition to ensure weighing accuracy. Next, 20 mg of disodium ferric ethylenediaminetetraacetate (EDTA) and organic components were added: 1000 mg inositol, 5 mg nicotinic acid, 0.5 mg pyridoxine hydrochloride, 5 mg thiamine hydrochloride, and 2 mg glycine. Finally, 30 g of sucrose was added as the carbon source.

[0044] Add 800 mL of double-distilled water to a beaker and stir on a magnetic stirrer at 300 rpm for 30 minutes, until all solid components are completely dissolved and the solution is clear and transparent. Then transfer the solution to a 1 L volumetric flask and dilute to the mark with double-distilled water. Invert the volumetric flask 3-5 times to ensure thorough mixing.

[0045] The pH value of the solution was measured using a calibrated pH meter. The pH was adjusted to 5.8 by adding 1M KOH solution dropwise. The solution was stirred continuously during the adjustment process to avoid local pH deviation. After each addition, the solution was allowed to stand for 30 seconds before measurement to ensure that the pH value was stable within the set range.

[0046] Transfer the adjusted basal culture medium to 250 mL sterile Erlenmeyer flasks using a sterile funnel, dispensing 100 mL into each flask. Seal the flasks with breathable sealing film and label them with the culture medium name, preparation date, and operator. Place the Erlenmeyer flasks in an autoclave and set the sterilization parameters to 121℃, 0.1 MPa, and 15 minutes. Closely monitor temperature and pressure changes during sterilization to ensure effective sterilization.

[0047] After sterilization, wait for the pressure in the sterilizer to drop naturally to atmospheric pressure and the temperature to drop below 80°C. Then, slowly open the exhaust valve, remove the triangular bottle, and place it in a clean bench. Cool it to room temperature (about 25°C) in a sterile environment for later use. Avoid touching the bottle opening during the cooling process to prevent contamination.

[0048] (III) Addition and preservation of functional components Plant growth regulators (NAA, TDZ), inducers (Dendrobium polysaccharide hydrolysate), and antioxidants (sodium selenate, glutathione) were prepared in advance into high-concentration stock solutions: NAA stock solution concentration was 100 mg / L, TDZ stock solution concentration was 50 mg / L, Dendrobium polysaccharide hydrolysate stock solution concentration was 1000 mg / L, sodium selenate stock solution concentration was 1 mM, and glutathione stock solution concentration was 1000 mg / L. All stock solutions were prepared with double-distilled water, filtered through a 0.22 μm sterile filter membrane for sterilization, and then dispensed into sterile centrifuge tubes and stored at -20℃ in the dark for later use.

[0049] In a clean bench, add the functional components to the basal culture medium cooled to room temperature: calculate the addition volume based on the stock solution concentration, and add 5 μL of NAA stock solution (final concentration 0.5 mg / L), 4 μL of TDZ stock solution (final concentration 0.2 mg / L), 1 mL of Dendrobium polysaccharide enzymatic hydrolysis product stock solution (final concentration 10 mg / L), 50 μL of sodium selenate stock solution (final concentration 5 μM), and 50 μL of glutathione stock solution (final concentration 5 mg / L) to 100 mL of basal culture medium.

[0050] After adding each component, gently invert the Erlenmeyer flask 5-6 times to ensure thorough mixing of the functional component with the basal medium, avoiding local concentrations that are too high or too low. After the addition is complete, measure the pH of the medium again to ensure it remains within the 5.7-5.9 range. If there is any deviation, it can be fine-tuned with a small amount of 1M KOH or 1M HCl solution.

[0051] Aliquot the prepared Dendrobium officinale stem cell vesicle-producing medium (MSH) into sterile centrifuge tubes or culture flasks, 20-50 mL per tube / flask. Seal the tubes / flasks, label with key information, and store at 4°C protected from light. During storage, check the medium weekly for signs of turbidity, precipitation, or microbial contamination. The shelf life is 30 days; medium exceeding this period should not be used. Before use, remove the medium from the refrigerator and allow it to reach room temperature for 30 minutes to avoid the impact of low temperature on stem cell growth.

[0052] Example 2 This embodiment provides a method for preparing Dendrobium stem cell extravesicles, including suspension culture of Dendrobium stem cells and extraction and purification of Dendrobium stem cell EVs (DSEVs).

[0053] Suspension culture of Dendrobium stem cells (I) Cell line selection and pretreatment Stem cell line acquisition (1) Preparation of sterile explants of Dendrobium: The Dendrobium pods were sterilized; (2) Callus induction by solid culture medium: Dendrobium sterile fruit pod powder was inoculated into solid culture medium and cultured in the dark at 25±2℃ for 15 days. After three subcultures, loose, yellowish-white Dendrobium callus with no obvious differentiation could be obtained.

[0054] The solid culture medium contains the following basic components: potassium nitrate 900 mg / L, ammonium nitrate 800 mg / L, potassium dihydrogen phosphate 85 mg / L, magnesium sulfate heptahydrate 185 mg / L, anhydrous calcium chloride 220 mg / L, ferrous sulfate 27.8 mg / L, disodium oxalate tetraacetate 37.3 mg / L, manganese sulfate 22.3 mg / L, boric acid 6.2 mg / L, 6-benzylaminopurine (concentration range 0–2.0 mg / L), potassium iodide 8.4 mg / L, sodium molybdate 0.26 mg / L, copper sulfate 0.026 mg / L, cobalt chloride 0.026 mg / L, inositol 100 mg / L, thioammonium hydrochloride 0.1 mg / L, nicotinic acid 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, glycine 0.2 mg / L, naphthaleneacetic acid 0.2 mg / L, and 2,4-dichlorophenoxyacetic acid 2.0 mg / L. Adjust the pH of the above basic component system to 5.8; then add 10 g / L mashed potatoes, 10 g / L mashed bananas, and 20 g / L sucrose, and stir thoroughly to ensure all components are evenly mixed. Prepare solutions in 100 mL increments per bottle, and add 1 g of agar powder per bottle. Sterilize at 121°C for 15 min. Meanwhile, this method is also applicable to the stem cell culture of other Dendrobium varieties such as Dendrobium huoshanense, Dendrobium nobile, and Dendrobium purpureus, requiring only a slight adjustment of the concentration of plant growth regulators in the culture medium according to the growth characteristics of different varieties of stem cells.

[0055] Cell pretreatment The induced Dendrobium callus tissue was inoculated at a rate of 30 g / L into Erlenmeyer flasks containing MSH culture medium containing Dendrobium stem cell vesicles produced in Example 1 for pre-culture. Pre-culture conditions included aeration of 10 L / min sterile air, low light conditions (below 500 lux), and culture at 25 ± 2°C and 110 rpm for 7 days. The aim was to allow the stem cells to adapt to the liquid suspension culture environment and restore their growth viability. After pre-culture, cell viability was assessed using the FDA / PI double staining method and observed under a fluorescence microscope. Live cells exhibited green fluorescence, while dead cells exhibited red fluorescence. Cells with a viability ≥90% were used for subsequent inoculation experiments.

[0056] (II) Experimental grouping and inoculation procedures The experiment was conducted with an experimental group and a control group, each with three biological replicates, each using a 250 mL sterile Erlenmeyer flask. The experimental group was given 100 mL of MSH medium prepared in Example 1 to produce Dendrobium stem cell vesicles, while the control group was given 100 mL of conventional MS medium (excluding Dendrobium polysaccharide hydrolysate, sodium selenate, and glutathione; all other components were the same as the experimental group). Both media were brought to room temperature and confirmed to be sterile beforehand.

[0057] Inoculation was performed in a clean bench: Select vigorous, uniformly morphologically developed Dendrobium stem cells in the logarithmic growth phase (days 7-10 of culture). Gently pipette the cell suspension in the culture flask to disperse the cells evenly and avoid large cell clusters. Then, using a sterile funnel and weighing paper, accurately weigh 10 g of fresh stem cells using the weight reduction method and inoculate them into a 250 mL Erlenmeyer flask containing 100 mL of culture medium, ensuring an inoculation volume of 100 g / L (fresh weight).

[0058] After inoculation, seal the Erlenmeyer flasks with breathable sealing film and gently invert them four times to ensure the stem cells are evenly distributed in the culture medium, preventing cells from settling to the bottom. Label each Erlenmeyer flask with the grouping information, inoculation date, and operator to ensure experimental traceability.

[0059] (III) Optimization and control of cultivation conditions After inoculation, the Erlenmeyer flasks were placed in a constant-temperature shaker, with the culture temperature set to 25±1℃. This temperature is the optimal temperature for the growth of Dendrobium stem cells. Temperatures that are too high (>28℃) will lead to decreased cell viability, while temperatures that are too low (<22℃) will inhibit cell proliferation. The shaker speed was set to 110 rpm. This speed ensures sufficient agitation of the culture medium, allowing the stem cells to receive adequate oxygen and nutrients, while avoiding cell damage caused by excessively high speeds.

[0060] The entire culture process is conducted in the dark, with the shaker wrapped in a black light-blocking cloth to prevent light from interfering with the photosynthesis of stem cells and the secretion of EVs. Dendrobium stem cells are more likely to maintain an undifferentiated state under dark conditions, and the yield and activity of EVs are higher.

[0061] The relative humidity of the culture environment is controlled at 50%-60%, achieved by placing a humidifier around the shaker. Suitable humidity prevents excessive evaporation of moisture from the culture medium and maintains stable osmotic pressure in the culture system. Simultaneously, the culture environment is disinfected regularly: the surface of the shaker is wiped daily with 75% ethanol, and ultraviolet disinfection is performed weekly to reduce the risk of contamination.

[0062] (iv) Monitoring and recording of the cultivation process Cell growth status monitoring: Samples were taken daily at a fixed time (9:00 AM) during the culture period, with a sample volume of 1 mL per group. Cell density was counted using a hemocytometer, and cell biomass was measured using both fresh weight and dry weight methods. Fresh weight measurement: After centrifuging the sample (1000×g, 5 minutes), the supernatant was removed, and the surface moisture of the stem cells was absorbed with sterile filter paper. The weight was then recorded. Dry weight measurement: The fresh weight sample was dried in a 60℃ oven until constant weight, cooled to room temperature, and then weighed. Cell growth curves were plotted based on the measurement results.

[0063] Culture system stability monitoring: After daily sampling, the pH value of the culture medium was measured using a pH meter. The normal range is 5.5-6.0. At the same time, the conductivity of the culture medium was measured using a conductivity meter. The initial conductivity was 2.5-3.0 mS / cm. As cells grow and nutrients are consumed, the conductivity gradually decreases to the normal level.

[0064] Cell viability monitoring: FDA / PI staining was performed every 3 days. 0.5 mL of cell suspension was taken, and FDA staining solution (final concentration 5 μg / mL) and PI staining solution (final concentration 10 μg / mL) were added. After incubation in the dark for 5 minutes, the cells were observed and counted under a fluorescence microscope, and cell viability was calculated (number of viable cells / total number of cells × 100%). Cell viability was ensured to be maintained above 85% during culture.

[0065] On days 0-30, collect all stem cells from each biological replicate. Collect cells by filtration using filter paper or microporous membrane of known weight. Rinse 2-3 times with deionized water to remove attached culture medium. Place the filter paper / membrane containing cells in an oven and dry at 60°C until constant weight (usually about 24-48 hours), then weigh using a precision balance. (Calculation formula: Dry weight of stem cells = Total weight after drying - Dry weight of filter paper / membrane). Figure 1 As shown, after 30 days of culture, the stem cell biomass (dry weight) in the experimental group increased by approximately 142.86% compared to the control group.

[0066] Extraction and purification of Dendrobium stem cell EVs (DSEVs) (a) Collection and pretreatment of supernatant After 14 days of culture, the preset culture cycle has been reached. Remove the Erlenmeyer flask from the shaker and place it in a clean bench. First, observe the state of the culture medium. Under normal circumstances, the culture medium should be a pale yellow, clear liquid, with cells uniformly suspended or slightly settled, and no obvious signs of turbidity or contamination.

[0067] Prepare a 400-mesh sterile nylon sieve, wipe it with 75% ethanol for disinfection, place a sterile centrifuge tube under the sieve, slowly pour the culture into the sieve, gently squeeze the cell clumps, and collect the clear filtrate. The purpose of this step is to remove undispersed cell clumps and large impurities to avoid affecting the subsequent centrifugation purification effect.

[0068] The collected filtrate was immediately transferred to a 4°C refrigerator for storage, with a refrigeration time not exceeding 2 hours, to prevent degradation or aggregation of extracellular vesicles due to prolonged exposure to room temperature. The filtrate volume was recorded as a basis for subsequent EV yield calculations.

[0069] (II) Differential centrifugation purification steps Primary centrifugation for cell removal: Transfer the refrigerated filtrate to a 50 mL sterile centrifuge tube, equilibrate, and place in a high-speed centrifuge. Set the centrifugation parameters to 4°C, 300×g, and centrifuge for 10 minutes. After centrifugation, carefully aspirate the supernatant to a new sterile centrifuge tube and discard the precipitate at the bottom of the tube (mainly residual intact cells). This step can remove more than 95% of free cells.

[0070] Secondary centrifugation to remove cell debris: Place the supernatant from the primary centrifugation into a high-speed centrifuge and set the parameters to 4℃, 2000×g, and centrifugation time for 20 minutes. During centrifugation, larger impurities such as cell debris and apoptotic bodies will settle to the bottom of the tube. After centrifugation, transfer the supernatant to a new centrifuge tube and discard the precipitate. At this point, the supernatant mainly contains EVs and a small amount of small molecule impurities.

[0071] Three-stage centrifugation to remove large vesicles: Transfer the supernatant from the secondary centrifugation to an ultracentrifuge tube. Set the ultracentrifuge parameters to 4°C, 10000×g, and centrifuge for 30 minutes. This step effectively removes impurities such as large vesicles and organelles (e.g., mitochondria, endoplasmic reticulum fragments), further purifying the supernatant containing EVs. After centrifugation, carefully aspirate the supernatant, avoiding contact with the precipitate at the bottom of the tube, and transfer the supernatant to a new ultracentrifuge tube for later use.

[0072] (III) Accumulation of EVs by ultracentrifugation Transfer the supernatant after differential centrifugation to Beckman Type 70 Ti ultracentrifuge tubes, with each tube's volume not exceeding 80% of the tube's maximum capacity. Use sterile PBS buffer to balance the weight of each tube, ensuring a weight deviation of ≤0.1 g to avoid instrument damage or affecting centrifugation results due to imbalance during centrifugation.

[0073] Place the equilibrated ultracentrifuge tubes into an ultracentrifuge and set the centrifugation parameters to 4℃, 100,000×g, and 70 minutes. During centrifugation, extracellular vesicles, due to their higher density (1.13-1.19 g / mL), will settle to the bottom of the tube, forming a white or pale yellow precipitate.

[0074] After centrifugation, slowly remove the centrifuge tube and carefully pour out the supernatant in a clean bench, removing as much residual liquid as possible to avoid washing away the precipitate. The precipitate at the bottom of the tube is the crude extract of Dendrobium stem cells (EVs), which needs to be purified immediately or briefly stored in a 4°C refrigerator (for no more than 1 hour).

[0075] (iv) Size exclusion chromatography purification and preservation Prepare a qEVoriginal 70 nm size size size size size size size size size column (IZON Science) in advance. Wash the column with sterile PBS buffer (pH 7.4) and equilibrate the column bed until the pH stabilizes at around 7.4. Set the flow rate to 0.5 mL / min and continue the equilibration process for 30 minutes to ensure the stability of the column performance.

[0076] The crude EVs extract obtained by ultracentrifugation was gently resuspended in 1 mL of sterile PBS buffer. During resuscitation, the EVs membrane structure was slowly broken down by pipetting with a pipette to avoid violent shaking. The number of pipetting cycles was controlled to 10-15 times until the precipitate was completely dissolved and a homogeneous suspension was formed.

[0077] Slowly load the resuspended solution onto the top of the column, avoiding the generation of air bubbles. After the suspension has completely entered the column bed, elute with sterile PBS buffer at a flow rate of 0.5 mL / tube. Collect the eluent sequentially, with a collection volume of 0.5 mL per tube, for a total of 20 tubes.

[0078] The particle concentration of the eluent in each tube was measured by NTA. The eluents from tubes 7-10, which had the highest particle concentration, were collected and combined to obtain high-purity Dendrobium officinale stem cell EVs (DSEVs). The purified DSEVs were transferred to sterile centrifuge tubes, sealed, and stored at -80°C, avoiding repeated freeze-thaw cycles during storage.

[0079] Transmission electron microscopy (TEM) showed that the EVs of Dendrobium stem cells were in good condition within the TEM field. Figure 2 The particle size distribution (NTA) of Dendrobium officinale stem cell EVs was further analyzed: 20 μL of purified DSEVs suspension was diluted 100-fold with sterile PBS buffer, thoroughly mixed, and added to the NTA sample cell. The detection parameters were set as follows: temperature 25℃, detection time 60 seconds, and lens focal length 15 mm. Each sample was tested three times, and the average value was taken. The results showed that the particle size of the Dendrobium officinale stem cell EVs in the experimental group was larger than that in the control group, indicating superior quality. Figure 3 ).

[0080] Example 3 Detection of activity indicators of Dendrobium stem cell EVs (DSEVs) (1) DPPH free radical scavenging ability: 1 mg of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) was dissolved in 20 mL of anhydrous ethanol and sonicated for 5 min. A0 value detection: 2 mL of the DPPH solution and 1 mL of anhydrous ethanol were thoroughly mixed and the absorbance was measured at 519 nm. A value detection: 2 mL of DPPH test solution was thoroughly mixed with 1 mL of gradient concentration and the absorbance was measured at 519 nm. AB value detection: 1.6 mL of DPPH test solution was mixed with 0.6 mL of water and the absorbance was measured at 734 nm. The experiment was independently repeated three times. Figure 4 ROS scavenging rate = (A0 - A+AB) / A0 * 100%. The results showed that the experimental group (Dendrobium officinale stem cells EVs prepared in MSH medium in Example 2) had a stronger DPPH free radical scavenging ability than the control group (Dendrobium officinale stem cells EVs prepared in conventional MS medium in Example 2).

[0081] (2) Cell scratch assay: Fibroblasts in the logarithmic growth phase were seeded at a concentration of 5 × 10⁶ cells / year. 5Cells were placed in 6-well plates, with 2.5 mL of culture medium per well, until the cells were evenly distributed. The 6-well plates were incubated in an incubator for 24 h. After 24 h, the supernatant was discarded, and the cells were washed twice. Using a 200 μL pipette tip, a blank scratch was quickly made perpendicular to the bottom of the 6-well plate along the cross mark. The same pipette tip was used for each well as much as possible. The cells were washed three times with PBS buffer to remove the scratched cells, and the scratches were checked under an inverted microscope to ensure they were clean. A PBS buffer control group and an experimental group (Dendrobium officinale stem cell vesicles prepared in Example 2) were set up. Fresh culture medium was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator. The results were recorded under a microscope at 0 h, 24 h, and 48 h. The scratch experiment results showed that the fibroblasts treated in the experimental group had a healing rate of about 60% higher than that in the control group at 24 h. Figure 5 ).

[0082] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for producing Dendrobium stem cell vesicles, characterized in that, include: 0.1-0.5 mg / L naphthaleneacetic acid, 0.05-0.2 mg / L thiamethoxam, 0.5-5 μM sodium selenate and 1-10 mg / L glutathione.

2. The culture medium for producing Dendrobium stem cell vesicles according to claim 1, characterized in that, The culture medium for producing Dendrobium stem cell vesicles also includes 1-10 mg / L of Dendrobium polysaccharide hydrolysis product, which is an oligosaccharide fragment obtained by hydrolyzing Dendrobium polysaccharide with cellulase or pectinase.

3. The culture medium for producing Dendrobium stem cell vesicles according to claim 1 or 2, characterized in that, The culture medium for producing Dendrobium stem cell vesicles includes: 0.2-0.5 mg / L naphthaleneacetic acid, 0.1-0.2 mg / L thidiazuron, 2-5 μM sodium selenate, 2-8 mg / L glutathione, and 5-10 mg / L Dendrobium polysaccharide hydrolysate.

4. The culture medium for producing Dendrobium stem cell vesicles according to claim 1 or 2, characterized in that, The dendrobium is selected from at least one of the following: Dendrobium officinale, Dendrobium huoshanense, Dendrobium nobile, Dendrobium purpureus, Dendrobium chrysanthum, Dendrobium chrysanthum, Dendrobium chrysanthum, and Dendrobium sphaeroides.

5. The culture medium for producing Dendrobium stem cell vesicles according to claim 1 or 2, characterized in that, The culture medium for producing Dendrobium stem cell vesicles also includes macro-elements, micro-elements, iron sources, organic components, and carbon sources.

6. A method for preparing Dendrobium stem cell vesicles, characterized in that, Dendrobium stem cells were cultured in suspension using the culture medium containing the outer vesicles of Dendrobium stem cells as described in any one of claims 1-5.

7. The method for preparing Dendrobium stem cell vesicles according to claim 6, characterized in that, The preparation method includes: Logarithmic growth phase Dendrobium stem cells were inoculated at 80-120 g / L (fresh weight) into the culture medium for producing Dendrobium stem cell vesicles as described in any one of claims 1-5, and cultured in the dark at 22-28℃ to obtain Dendrobium stem cell vesicles.

8. The method for preparing Dendrobium stem cell vesicles according to claim 7, characterized in that, The method for culturing Dendrobium stem cells includes: inoculating Dendrobium callus at 20-40 g / L (fresh weight) into the culture medium containing Dendrobium stem cell-producing vesicles as described in any one of claims 1-4, introducing sterile air at a rate of 5-10 L / min, culturing at a low light level below 500 lux and at 23-27°C for 15-45 days, and obtaining Dendrobium stem cells through 3-5 subcultures.

9. A type of Dendrobium stem cell vesicle, characterized in that, It is prepared by the method for preparing Dendrobium stem cell vesicles according to any one of claims 6 to 8.

10. The use of Dendrobium stem cell vesicles as described in claim 9 in the preparation of cosmetics or pharmaceuticals with skin repair, anti-inflammatory, or immunomodulatory functions.

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