A deer placental stem cell exosome and its preparation method

Abstract

This invention relates to the field of tissue engineering technology, specifically disclosing a deer placental stem cell exosome and its preparation method. The invention uses deer placental chorionic villus tissue as raw material, and obtains highly active stem cells through mild enzymatic hydrolysis, differential adhesion, and purification in a metabolically stable culture medium. Serum-free stress induction is performed using the mitochondrial uncoupling agent FCCP, combined with differential centrifugation, double-layer filtration, gradient density centrifugation, and ultrafiltration concentration to achieve efficient purification. Stable exosomes are obtained through vitamin E polyethylene glycol succinate-soybean lecithin complex modification. The preparation process of this invention is mild, scalable, and the resulting exosomes exhibit strong antioxidant and cell proliferation-promoting abilities, high stability, and good bioavailability, making them suitable for tissue regeneration and repair, anti-inflammatory and anti-aging applications, skin care, and stem cell culture additives.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering technology, specifically to a deer placental stem cell exosome and its preparation method. Background Technology

[0002] Deer placenta, serving as a nutrient exchange and immune regulation organ in the later stages of pregnancy in deer, is rich in highly active mesenchymal stem cells, various growth factors, functional peptides, and antioxidant active ingredients. It possesses excellent tissue repair, anti-inflammatory, antioxidant, and cell proliferation-promoting capabilities, making it a high-quality natural source for regenerative medicine and bioactive raw materials. Deer placental stem cell exosomes, as the core functional carriers of stem cell paracrine function, can carry specific miRNAs, active proteins, and lipid molecules, demonstrating great application potential in cell-free therapy, skin anti-aging, and wound repair.

[0003] While significant progress has been made in stem cell exosome preparation technology, substantial technical bottlenecks remain in the field of deer placental stem cell exosomes. Firstly, cell isolation and stemness maintenance processes are relatively rudimentary, often relying on conventional enzymatic digestion and adherent culture. This results in insufficient stem cell purity, easy degradation of activity, and difficulty in consistently obtaining highly homogeneous seed cells, directly leading to large batch-to-batch variations and unstable exosome activity. Secondly, exosome secretion induction methods are limited, often relying on hypoxic culture or serum supplementation, failing to achieve efficient enrichment of active components. Consequently, exosome yield and bioactivity cannot meet the requirements for industrial applications. Thirdly, purification processes often employ ultracentrifugation, density gradient centrifugation, or antibody affinity purification, which are cumbersome, prone to exosome structural damage, have low recovery rates, and are costly, hindering large-scale production. Fourthly, conventional exosomes lack in vivo stabilization modifications, making them susceptible to degradation and clearance during storage and in vivo delivery. Their short half-life and low bioavailability severely limit their clinical and commercial applications.

[0004] Based on the above statements, this invention proposes a deer placental stem cell exosome and its preparation method. Summary of the Invention

[0005] To address the problems of low purity, insufficient yield, unstable activity, difficulty in large-scale production, easy in vivo inactivation, and low bioavailability in the preparation of deer placental stem cell exosomes, this invention proposes a deer placental stem cell exosome and its preparation method.

[0006] In a first aspect, the present invention provides a method for preparing deer placental stem cell exosomes, employing the following technical solution: A method for preparing deer placental stem cell exosomes includes the following steps: S1. Fresh placenta was collected from healthy pregnant does. The placental chorion tissue was aseptically peeled off, washed, and mechanically cut to obtain tissue fragments. The tissue fragments were enzymatically digested with a mild enzymatic digestion solution and examined under a microscope in real time until the cells were fully freed. Digestion was terminated, centrifuged, the supernatant was discarded, the cell pellet was washed, and the cell pellet was resuspended in deer placental stem cell basal culture medium. The cells were incubated, and the non-adherent cells and culture medium were discarded. The adherent cells were collected and seeded in a purified culture medium for culture and screening to obtain deer placental stem cells. S2. Deer placental stem cells were seeded into cell culture flasks, serum-free induction medium was added, mitochondrial uncoupling agent was added, and induction culture was performed. After induction, the culture supernatant was collected, centrifuged, and the supernatant to be purified was obtained. S3. The supernatant to be purified is centrifuged by differential speed, filtered in two layers, centrifuged by gradient density and concentrated by ultrafiltration to obtain the deer placental stem cell exosome stock solution. S4. The deer placental stem cell exosome stock solution was mixed with a double-modification reagent and incubated to obtain deer placental stem cell exosomes.

[0007] Preferably, the mild enzymatic hydrolysate in step S1 consists of 10-20 U / mL collagenase IV and 0.03-0.08 wt% trypsin inhibitor.

[0008] Preferably, the basal culture medium for deer placental stem cells in step S1 is α-MEM medium containing 8-15% (v / v) exosome-free fetal bovine serum, 90-110 U / mL penicillin, and 90-110 μg / mL streptomycin.

[0009] Preferably, the purification medium in step S1 is α-MEM medium containing 50-150 μmol / L L-carnitine and 100-300 μmol / L taurine.

[0010] Preferably, the incubation conditions in step S1 are: incubation at 35-40℃ and 4-6% CO2 for 45-75 minutes.

[0011] Preferably, the culture and screening in step S1 is as follows: culture until the cell confluence is ≥80%, select cells with uniform morphology and good growth status for passage expansion, and passage to P2-P4 generation.

[0012] Preferably, the serum-free induction medium in step S2 is α-MEM medium containing 2-5% (w / v) bovine serum albumin, 90-110 U / mL penicillin, 90-110 μg / mL streptomycin, 10-50 μg / mL vitamin C, and 3-5 μg / mL insulin.

[0013] Preferably, the mitochondrial uncoupling agent in step S2 is FCCP, and the final concentration added is 5-20 nmol / L.

[0014] Preferably, the induction culture conditions in step S2 are: induction culture at 35-40℃ and 4-6% CO2 for 24-48 hours.

[0015] Preferably, the differential centrifugation in step S3 includes: centrifuging at 1000-1500×g for 8-12 min, centrifuging at 3000-5000×g for 15-25 min, and centrifuging at 10000-13000×g for 25-35 min at 4-8℃.

[0016] Preferably, the dual-modification reagent in step S4 is composed of vitamin E polyethylene glycol succinate and soybean lecithin in a mass ratio of 2-4:1.

[0017] Preferably, the mixed incubation conditions in step S4 are: incubation at 25-35℃ for 40-60 minutes.

[0018] Secondly, the present invention provides a method for preparing deer placental stem cell exosomes and the deer placental stem cell exosomes obtained therefrom.

[0019] Thirdly, this invention provides the application of deer placental stem cell exosomes in the preparation of tissue regeneration and repair agents, anti-inflammatory and anti-aging agents, skin care raw materials, targeted delivery carriers, and stem cell culture additives.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Significantly improved stem cell purity and activity, and stronger batch stability: This invention uses mild enzymatic hydrolysis combined with differential adhesion purification, and is cultured in a purification medium containing L-carnitine and taurine. This can efficiently maintain the stemness and metabolic homeostasis of deer placental stem cells, and obtain high-purity, high-viability seed cells. No complex sorting equipment is required throughout the process, the cells are uniform in morphology and have stable proliferation, reducing batch differences in exosomes from the source and ensuring stable and controllable product quality.

[0021] (2) Exosome secretion efficiency is greatly improved and active ingredients are more fully enriched: The use of low-concentration mitochondrial uncoupling agents for mild stress induction significantly activates the exosome synthesis and secretion pathways without damaging cells, resulting in a significant increase in exosome production compared to conventional culture methods. At the same time, the use of a serum-free induction system with well-defined chemical components effectively enriches repair, antioxidant, and anti-inflammatory active substances, thereby significantly enhancing the biological activity of exosomes.

[0022] (3) The purification process is mild and efficient, meeting the needs of industrial scale-up: This invention adopts a purification route combining differential centrifugation, double-layer filtration, gradient density centrifugation, and ultrafiltration concentration. The entire operation is mild and does not damage the phospholipid bilayer structure of exosomes, which can efficiently remove impurity proteins and cell debris to obtain high-purity exosomes. The process route is simple, low-cost, and has good reproducibility, without the need for expensive equipment such as ultracentrifugation and antibody affinity testing.

[0023] (4) Significantly improved exosome stability, making storage and application more convenient: The composite modification of vitamin E polyethylene glycol succinate and soybean lecithin forms a stable protective layer on the vesicle surface, significantly improving the exosome's resistance to degradation, freeze-thaw cycles, and shear stress, effectively reducing activity loss during room temperature storage and transportation. Compared with unmodified exosomes, it has stronger stability and a longer shelf life, significantly reducing reliance on cold chain and application costs.

[0024] (5) Wider range of applications and outstanding industrialization and application value: The deer placental stem cell exosomes prepared by this invention have high activity, high stability and high biocompatibility, and can be widely used in multiple fields such as tissue regeneration and repair, anti-inflammatory and anti-aging, skin care, targeted delivery, and stem cell culture addition. The overall process is safe and controllable, free of serum and animal-derived contamination risks, meets the application requirements of biomedical and medical aesthetic raw materials, and has outstanding market transformation and industrialization prospects. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0026] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0027] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0028] The PBS buffer was purchased from Yisheng Biotechnology, product code: 41403ES76. Collagenase IV was purchased from Yisheng Biotechnology, product code: 40510ES60; The trypsin inhibitor was purchased from Yisheng Biotechnology, product code: 733485ES50; Exosome-free fetal bovine serum was purchased from Yisheng Biotechnology, product code: 40139ES50; α-MEM medium was purchased from Thermo Fisher, product code: 12561056; The mitochondrial uncoupling agent FCCP was purchased from Wuhan Sanying Biotechnology Co., Ltd., product code: CM00253; Vitamin E polyethylene glycol succinate was purchased from Ruixi Biotechnology, product code: R-V819469-2k; Soybean lecithin was purchased from Shaanxi Xintianyu Biotechnology Co., Ltd.

[0029] Example 1 A method for preparing deer placental stem cell exosomes includes the following steps: S1. Isolation and purification of deer placental stem cells: Fresh placentas were collected from healthy pregnant does. The placental chorionic membrane tissue was aseptically detached, washed with PBS buffer until no blood remained, and mechanically cut into 0.4 mm pieces. 3 Tissue fragments were collected; the tissue fragments were mixed with mild enzymatic digestion solution (composed of 10 U / mL collagenase IV and 0.03 wt% trypsin inhibitor) at a mass ratio of 1:3, and enzymatically digested at 35°C for 15 min. Cells were observed under a microscope until fully freed. Digestion was terminated by adding an equal volume of α-MEM medium containing 8% (v / v) exosome-free fetal bovine serum. The cells were centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the precipitate was washed twice with PBS buffer. The cell pellet was resuspended in deer placental stem cell basal medium (α-MEM medium containing 8% (v / v) exosome-free fetal bovine serum, 90 U / mL penicillin, and 90 μg / mL streptomycin), and the cell suspension concentration was adjusted to 1 × 10⁻⁶. 5 Cells / mL were incubated at 35°C and 4% CO2 for 45 min. Unattached cells and culture medium were discarded, and adherent cells were collected and cultured at 1×10⁻⁶. 4 Inoculate cells at a rate of 1 cell / cm² into purified medium (α-MEM medium containing 50 μmol / L L-carnitine and 100 μmol / L taurine) and continue culturing; when the cell confluence reaches 80%, select cells with uniform morphology and good growth status for passage expansion, passage to P2 generation to obtain deer placental stem cells. S2. Induction culture and exosome secretion of deer placental stem cells: Deer placental stem cells were used at a rate of 1×10 5Cells were seeded at a density of 10 cells / mL in cell culture flasks and inoculated with serum-free induction medium (α-MEM medium containing 2% (w / v) bovine serum albumin, 90 U / mL penicillin, 90 μg / mL streptomycin, 10 μg / mL vitamin C, and 3 μg / mL insulin). Mitochondrial uncoupling agent FCCP was added to the serum-free induction medium to a final concentration of 5 nmol / L. The cells were then induced and cultured at 35°C and 4% CO2 for 24 h. The culture supernatant was collected, centrifuged at 3000 rpm for 8 min, and cell debris was removed to obtain the supernatant to be purified. S3. Extraction and purification of deer placental stem cell exosomes: The supernatant to be purified was placed at 4°C and centrifuged sequentially at 1000×g for 8 min, 3000×g for 15 min, and 10000×g for 25 min. It was then filtered through a double-layer filter membrane consisting of a 0.45 μm filter and a 0.22 μm filter membrane. The filtered supernatant was transferred to an ultracentrifuge tube for density gradient centrifugation. The ultracentrifuge tube contained, from bottom to top, 60% sucrose solution, 30% sucrose solution, and the filtered supernatant. The tube was centrifuged at 4°C and 120000×g for 75 min. After centrifugation, enriched exosomes were obtained. The enriched exosomes were diluted with PBS buffer at a volume ratio of 1:4 and transferred to a 300 kDa ultrafiltration tube. The solution was concentrated to 1 / 10 of its original volume by centrifugation at 4°C and 4000×g to obtain the deer placental stem cell exosome stock solution. S4. Encapsulation modification of deer placental stem cell exosomes: The deer placental stem cell exosome stock solution was mixed with a dual-modification reagent (composed of vitamin E polyethylene glycol succinate and soybean lecithin in a mass ratio of 2:1) at a volume ratio of 1:1 and incubated at 25°C for 40-60 min to obtain deer placental stem cell exosomes.

[0030] Example 2 A method for preparing deer placental stem cell exosomes includes the following steps: S1. Isolation and purification of deer placental stem cells: Fresh placentas were collected from healthy pregnant does. The placental chorionic membrane tissue was aseptically detached, washed with PBS buffer until no blood remained, and mechanically cut into 0.5 mm pieces. 3Tissue fragments were collected; the tissue fragments were mixed with mild enzymatic digestion solution (composed of 15 U / mL collagenase IV and 0.05 wt% trypsin inhibitor) at a mass ratio of 1:4, and enzymatically digested at 37°C for 20 min. Cells were observed under a microscope until fully freed. Digestion was terminated by adding an equal volume of α-MEM medium containing 10% (v / v) exosome-free fetal bovine serum. The cells were centrifuged at 1000 rpm for 6 min, the supernatant was discarded, and the pellet was washed twice with PBS buffer. The cell pellet was resuspended in deer placental stem cell basal medium (α-MEM medium containing 10% (v / v) exosome-free fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin), and the cell suspension concentration was adjusted to 3 × 10⁻⁶. 5 Cells / mL were incubated at 37°C and 5% CO2 for 60 min. Unattached cells and culture medium were discarded, and adherent cells were collected and cultured at 3×10⁻⁶. 4 Inoculate cells at a rate of 1 cell / cm² into purified medium (α-MEM medium containing 100 μmol / L L-carnitine and 200 μmol / L taurine) and continue culturing; when the cell confluence reaches 80%, select cells with uniform morphology and good growth status for passage expansion, passage to P3, and obtain deer placental stem cells. S2. Induction culture and exosome secretion of deer placental stem cells: Deer placental stem cells were used at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of cells / mL in cell culture flasks and added to serum-free induction medium (α-MEM medium containing 3% (w / v) bovine serum albumin, 100 U / mL penicillin, 100 μg / mL streptomycin, 30 μg / mL vitamin C, and 4 μg / mL insulin). Mitochondrial uncoupling agent FCCP was added to the serum-free induction medium to a final concentration of 10 nmol / L. The cells were then induced and cultured at 37°C and 5% CO2 for 36 h. The culture supernatant was collected, centrifuged at 3500 rpm for 10 min, and cell debris was removed to obtain the supernatant to be purified. S3. Extraction and purification of deer placental stem cell exosomes: The supernatant to be purified was placed at 6°C and centrifuged sequentially at 1200×g for 10 min, 4000×g for 20 min, and 12000×g for 30 min. It was then filtered through a double-layer filter membrane consisting of a 0.45 μm filter and a 0.22 μm filter membrane. The filtered supernatant was transferred to an ultracentrifuge tube for density gradient centrifugation. The ultracentrifuge tube contained, from bottom to top, 60% sucrose solution, 30% sucrose solution, and the filtered supernatant. The tube was centrifuged at 4°C and 120000×g for 75 min. After centrifugation, enriched exosomes were obtained. The enriched exosomes were diluted with PBS buffer at a volume ratio of 1:4 and transferred to a 300 kDa ultrafiltration tube. The solution was concentrated to 1 / 10 of its original volume by centrifugation at 4°C and 4000×g to obtain the deer placental stem cell exosome stock solution. S4. Encapsulation modification of deer placental stem cell exosomes: The deer placental stem cell exosome stock solution was mixed with a dual-modification reagent (composed of vitamin E polyethylene glycol succinate and soybean lecithin in a mass ratio of 3:1) at a volume ratio of 1:1.2 and incubated at 30°C for 50 min to obtain deer placental stem cell exosomes.

[0031] Example 3 A method for preparing deer placental stem cell exosomes includes the following steps: S1. Isolation and purification of deer placental stem cells: Fresh placentas were collected from healthy pregnant does. The placental chorionic membrane tissue was aseptically detached, washed with PBS buffer until no blood remained, and mechanically cut into 0.6 mm pieces. 3 Tissue fragments were collected; the tissue fragments were mixed with mild enzymatic digestion solution (composed of 20 U / mL collagenase IV and 0.08 wt% trypsin inhibitor) at a mass ratio of 1:5, and enzymatically digested at 40℃ for 25 min. Cells were observed under a microscope until fully freed. Digestion was terminated by adding an equal volume of α-MEM medium containing 12% (v / v) exosome-free fetal bovine serum. The cells were centrifuged at 1200 rpm for 8 min, the supernatant was discarded, and the pellet was washed three times with PBS buffer. The cell pellet was resuspended in deer placental stem cell basal medium (α-MEM medium containing 15% (v / v) exosome-free fetal bovine serum, 110 U / mL penicillin, and 110 μg / mL streptomycin), and the cell suspension concentration was adjusted to 5 × 10⁻⁶ cells / mL. 5 Cells / mL were incubated at 40℃ and 6% CO2 for 75 min. Unattached cells and culture medium were discarded, and adherent cells were collected and cultured at 5×10⁻⁶. 4Inoculate cells at a rate of 1 cell / cm² into purified medium (α-MEM medium containing 150 μmol / L L-carnitine and 300 μmol / L taurine) and continue culturing; when the cell confluence reaches 80%, select cells with uniform morphology and good growth status for passage expansion, passage to P4, and obtain deer placental stem cells. S2. Induction culture and exosome secretion of deer placental stem cells: Deer placental stem cells were used at a rate of 2×10 5 Cells were seeded at a density of 10 cells / mL in cell culture flasks and added to serum-free induction medium (α-MEM medium containing 5% (w / v) bovine serum albumin, 110 U / mL penicillin, 110 μg / mL streptomycin, 50 μg / mL vitamin C, and 5 μg / mL insulin). Mitochondrial uncoupling agent FCCP was added to the serum-free induction medium to a final concentration of 20 nmol / L. The cells were then induced and cultured at 40℃ and 6% CO2 for 48 h. The culture supernatant was collected, centrifuged at 4000 rpm for 12 min, and cell debris was removed to obtain the supernatant to be purified. S3. Extraction and purification of deer placental stem cell exosomes: The supernatant to be purified was placed at 8°C and centrifuged sequentially at 1500×g for 12 min, 5000×g for 25 min, and 13000×g for 35 min. It was then filtered through a double-layer filter membrane consisting of a 0.45 μm filter and a 0.22 μm filter membrane. The filtered supernatant was transferred to an ultracentrifuge tube for density gradient centrifugation. The ultracentrifuge tube contained, from bottom to top, 60% sucrose solution, 30% sucrose solution, and the filtered supernatant. Centrifugation was performed at 4°C and 120000×g for 75 min. The resulting enriched exosome fraction was then obtained. This enriched exosome fraction was diluted with PBS buffer at a volume ratio of 1:4 and transferred to a 300 kDa ultrafiltration tube. The solution was concentrated to 1 / 10 of its original volume by centrifugation at 4°C and 4000×g to obtain the deer placental stem cell exosome stock solution. S4. Encapsulation modification of deer placental stem cell exosomes: The deer placental stem cell exosome stock solution was mixed with a dual-modification reagent (composed of vitamin E polyethylene glycol succinate and soybean lecithin in a mass ratio of 4:1) at a volume ratio of 1:1.5 and incubated at 35°C for 60 min to obtain deer placental stem cell exosomes.

[0032] Comparative Example 1 The only difference between this comparative example and Example 2 is that the purification culture and screening step in step S1 is omitted: the adherent cells are directly cultured in the basic culture medium of deer placental stem cells, and the α-MEM purification culture medium containing L-carnitine and taurine is not used. The other raw material ratios, preparation processes and all parameters are completely consistent with Example 2.

[0033] Specifically: S1. Isolation and purification of deer placental stem cells: Fresh placentas were collected from healthy pregnant does. The placental chorionic membrane tissue was aseptically detached, washed with PBS buffer until no blood remained, and mechanically cut into 0.5 mm pieces. 3 Tissue fragments were collected; the tissue fragments were mixed with mild enzymatic digestion solution (composed of 15 U / mL collagenase IV and 0.05 wt% trypsin inhibitor) at a mass ratio of 1:4, and enzymatically digested at 37°C for 20 min. Cells were observed under a microscope until fully freed. Digestion was terminated by adding an equal volume of α-MEM medium containing 10% (v / v) exosome-free fetal bovine serum. The cells were centrifuged at 1000 rpm for 6 min, the supernatant was discarded, and the pellet was washed twice with PBS buffer. The cell pellet was resuspended in deer placental stem cell basal medium (α-MEM medium containing 10% (v / v) exosome-free fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin), and the cell suspension concentration was adjusted to 3 × 10⁻⁶. 5 Cells / mL were incubated at 37°C and 5% CO2 for 60 min. Unattached cells and culture medium were discarded, and adherent cells were collected and cultured at 3×10⁻⁶. 4 Inoculate the cells at a rate of 1 cell / cm² into the basal culture medium of deer placental stem cells and continue culturing. When the cell confluence reaches 80%, select cells with uniform morphology and good growth status for passage expansion. Passage to the P3 generation to obtain deer placental stem cells.

[0034] Comparative Example 2 The only difference between this comparative example and Example 2 is that the mitochondrial uncoupling agent FCCP is not added in step S2. The remaining raw material ratios, preparation processes and all parameters are completely consistent with Example 2.

[0035] Specifically: S2. Induction culture and exosome secretion of deer placental stem cells: Deer placental stem cells were used at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of cells / mL in cell culture flasks and added to serum-free induction medium (α-MEM medium containing 3% (w / v) bovine serum albumin, 100 U / mL penicillin, 100 μg / mL streptomycin, 30 μg / mL vitamin C, and 4 μg / mL insulin). The cells were then incubated at 37°C and 5% CO2 for 36 h. The culture supernatant was collected, centrifuged at 3500 rpm for 10 min, and cell debris was removed to obtain the supernatant to be purified.

[0036] Comparative Example 3 The only difference between this comparative example and Example 2 is that the dual-modifying reagent in step S4 is replaced by vitamin E polyethylene glycol succinate in equal mass. The remaining raw material ratios, preparation processes and all parameters are completely consistent with Example 2.

[0037] Specifically: S4. Encapsulation modification of deer placental stem cell exosomes: Deer placental stem cell exosome stock solution was mixed with vitamin E polyethylene glycol succinate at a volume ratio of 1:1.2 and incubated at 30℃ for 50 min to obtain deer placental stem cell exosomes.

[0038] Comparative Example 4 The only difference between this comparative example and Example 2 is that the dual-modifying reagent in step S4 is replaced by soybean lecithin by the same mass. The other raw material ratios, preparation processes and all parameters are completely consistent with Example 2.

[0039] Specifically: S4. Encapsulation modification of deer placental stem cell exosomes: The deer placental stem cell exosome stock solution was mixed with soybean lecithin at a volume ratio of 1:1.2 and incubated at 30℃ for 50 min to obtain deer placental stem cell exosomes.

[0040] Performance testing 1. Testing Method Deer placental stem cell exosomes prepared in Examples 1-3 and Comparative Examples 1-4 were resuspended in 0.01 mol / L PBS buffer at pH 7.4 to the same protein concentration (0.5 mg / mL), aliquoted into 100 μL / tube, stored at 4°C in the dark, and tested within 24 hours. All operations were performed under sterile conditions at room temperature (25°C). (1) DPPH free radical scavenging rate test (antioxidant capacity): Accurately weigh the DPPH reagent, dissolve it in anhydrous ethanol and dilute to volume to prepare a 0.1 mmol / L DPPH anhydrous ethanol solution. Prepare and use immediately, and store in a dark place on a work surface after preparation. Use sterile, light-protected centrifuge tubes to set up sample groups, sample control groups, and blank control groups. Each group should have 3 technical replicates and 3 parallel duplicates. The sample volumes are as follows: Sample group: 10 μL of 0.5 mg / mL deer placental stem cell exosome sample prepared in Examples 1-3 and Comparative Examples 1-4 + 1000 μL of 0.1 mmol / L DPPH anhydrous ethanol solution, gently vortex to mix. Sample control group: 10 μL of 0.5 mg / mL deer placental stem cell exosome samples prepared in Examples 1-3 and Comparative Examples 1-4 + 1000 μL of anhydrous ethanol, gently vortex to mix (to eliminate interference from the sample's own absorbance). Blank control group: 10 μL of 0.01 mol / L PBS buffer at pH 7.4 + 1000 μL of 0.1 mmol / L DPPH anhydrous ethanol solution, gently vortex to mix (as a reference for the initial absorbance of DPPH free radicals). After adding the sample, let the centrifuge tube stand at room temperature in the dark for 30 minutes to react, avoiding shaking during this time; After the reaction is complete, 200 μL of the reaction solution is transferred from each centrifuge tube to a sterile 96-well microplate (one duplicate tube per well). The microplate reader is set to 519 nm wavelength and the zero point is calibrated. The absorbance (A) of each well is measured in sequence and the data is recorded. During the measurement, ensure that there are no air bubbles on the microplate and no droplets on the well walls. Calculation formula: Clearance rate (%) = [1 - (A)] 样品组 -A 样品对照组 ) / A 空白对照组 ×100%; In the formula: A 样品组 A represents the average absorbance of the sample group. 样品对照组 A represents the average absorbance of the sample and control groups. 空白对照组 The absorbance is the average value of the blank control group.

[0041] (2) Cell proliferation promotion test (regeneration and repair capacity): This test simultaneously detected HaCaT cells and NHDF cells, using identical procedures. Independent experimental and control groups were set up for each group, as detailed below: Cell seeding: HaCaT and NHDF cells in logarithmic growth phase were digested with 0.25% (w / v) trypsin, resuspended in DMEM medium containing 10% fetal bovine serum, and the cell density was adjusted to 5 × 10⁶ cells / year. 3 For each well, take a sterile, transparent 96-well microplate, add 100 μL of cell suspension to each well, and set up 3 technical replicates and 3 parallel replicates for each group; Adherent cell culture: Place the 96-well plate in a constant temperature cell culture incubator at 37℃, 5% CO2, and saturated humidity, and culture for 24 hours until the cell adhesion rate reaches 80%. Sample preparation: Discard the original culture medium in the 96-well plate, wash the cells twice with 0.01 mol / L PBS buffer (pH 7.4), and discard the washing solution; then add α-MEM medium containing exosome samples to achieve a final exosome protein concentration of 50 μg / mL, with a sample volume of 100 μL per well; at the same time, a blank control group was set up, with an equal volume of α-MEM medium added (without exosome samples). Co-culture: After adding the samples, place the 96-well plate back into the constant temperature cell culture incubator and co-culture at 37℃ and 5% CO2 for 24 hours. Avoid shaking the incubator during this period. CCK-8 incubation: After co-culture, add 10 μL of CCK-8 detection reagent to each well (avoid generating air bubbles), gently shake the 96-well plate to mix the reagent, and continue to incubate in an incubator at 37°C for 2 hours; Absorbance measurement: After incubation, the microplate reader was set to 450 nm wavelength, with 630 nm as the reference wavelength. After calibrating the zero point, the absorbance value (A) of each well was measured and the data was recorded. Calculation formula: Cell proliferation rate (%) = (A 样品组 / A 空白对照组 )×100%; In the formula: A 样品组 A represents the average absorbance of the cell pores in the exosome sample. 空白对照组 The values ​​represent the average absorbance of cell pores without exosomes.

[0042] 2. Test Results The specific test results are shown in Table 1.

[0043] Table 1. Performance test results of deer placental stem cell exosomes As shown in Table 1, the deer placental stem cell exosomes prepared in Examples 1-3 of this invention all have excellent in vitro antioxidant capacity and cell proliferation promotion effect, among which Example 2 has the best performance.

[0044] Comparing Example 2 with Comparative Example 1, it can be seen that Comparative Example 1 omitted the purification culture medium screening step S1, resulting in a decrease in DPPH free radical scavenging rate and a significant reduction in cell proliferation rate. Its performance was the worst among all samples, indicating that the screening culture of the purified culture medium is a key step in obtaining highly active deer placental stem cells and thereby enhancing the core efficacy of exosomes.

[0045] As can be seen from the comparison between Example 2 and Comparative Example 2, Comparative Example 2 did not add the mitochondrial uncoupling agent FCCP in step S2. The antioxidant capacity and cell proliferation promotion effect of exosomes were significantly reduced, and all indicators were lower than those of Example 2. This shows that the induction and stimulation of FCCP can effectively activate the stress secretion mechanism of deer placental stem cells and significantly increase the content of active factors in exosomes. It is one of the core processes to achieve high exosome activity.

[0046] A comparison of Example 2 and Comparative Example 3 shows that: Comparative Example 3 only used a single vitamin E polyethylene glycol succinate for encapsulation modification, replacing the vitamin E polyethylene glycol succinate-soybean lecithin composite carrier. The exosome performance was significantly reduced, indicating that the composite carrier synergistic encapsulation can form a more stable protective layer on the surface of exosomes, reduce the loss of active ingredients, and significantly improve the structural stability and biological activity of exosomes.

[0047] A comparison between Example 2 and Comparative Example 4 shows that Comparative Example 4 uses only soybean lecithin for encapsulation modification, replacing the vitamin E polyethylene glycol succinate-soybean lecithin composite carrier. However, the performance indicators of exosomes are still lower than those of Example 2, indicating that vitamin E polyethylene glycol succinate and soybean lecithin have a significant synergistic effect. The composite encapsulation of the two can more effectively maintain the integrity of exosomes. The absence of either component will cause the overall performance of exosomes to fail to reach the optimal state.

[0048] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for preparing deer placental stem cell exosomes, characterized in that, Includes the following steps: S1. Fresh placenta was collected from healthy pregnant does. The placental chorion tissue was aseptically peeled off, washed, and mechanically cut to obtain tissue fragments. The tissue fragments were enzymatically digested with a mild enzymatic digestion solution and examined under a microscope in real time until the cells were fully freed. Digestion was terminated, centrifuged, the supernatant was discarded, the cell pellet was washed, and the cell pellet was resuspended in deer placental stem cell basal culture medium. The cells were incubated, and the non-adherent cells and culture medium were discarded. The adherent cells were collected and seeded in a purified culture medium for culture and screening to obtain deer placental stem cells. S2. Deer placental stem cells were seeded into cell culture flasks, serum-free induction medium was added, mitochondrial uncoupling agent was added, and induction culture was performed. After induction, the culture supernatant was collected, centrifuged, and the supernatant to be purified was obtained. S3. The supernatant to be purified is centrifuged by differential speed, filtered in two layers, centrifuged by gradient density and concentrated by ultrafiltration to obtain the deer placental stem cell exosome stock solution. S4. The deer placental stem cell exosome stock solution was mixed with a double-modification reagent and incubated to obtain deer placental stem cell exosomes.

2. The method for preparing deer placental stem cell exosomes according to claim 1, characterized in that, In step S1, the mild enzymatic hydrolysate consists of 10-20 U / mL collagenase IV and 0.03-0.08 wt% trypsin inhibitor.

3. The method for preparing deer placental stem cell exosomes according to claim 1, characterized in that, In step S1, the basic culture medium for deer placental stem cells is α-MEM medium containing 8-15% (v / v) exosome-free fetal bovine serum, 90-110 U / mL penicillin, and 90-110 μg / mL streptomycin.

4. The method for preparing deer placental stem cell exosomes according to claim 1, characterized in that, The purification medium in step S1 is α-MEM medium containing 50-150 μmol / L L-carnitine and 100-300 μmol / L taurine.

5. The method for preparing deer placental stem cell exosomes according to claim 1, characterized in that, In step S2, the serum-free induction medium is α-MEM medium containing 2-5% (w / v) bovine serum albumin, 90-110 U / mL penicillin, 90-110 μg / mL streptomycin, 10-50 μg / mL vitamin C, and 3-5 μg / mL insulin.

6. The method for preparing deer placental stem cell exosomes according to claim 1, characterized in that, In step S2, the mitochondrial uncoupling agent is FCCP, and the final concentration added is 5-20 nmol / L.

7. The method for preparing deer placental stem cell exosomes according to claim 1, characterized in that, The differential centrifugation in step S3 includes: centrifuging at 1000-1500×g for 8-12 min, 3000-5000×g for 15-25 min, and 10000-13000×g for 25-35 min at 4-8℃.

8. The method for preparing deer placental stem cell exosomes according to claim 1, characterized in that, In step S4, the dual-modification reagent is composed of vitamin E polyethylene glycol succinate and soybean lecithin in a mass ratio of 2-4:

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9. Deer placental stem cell exosomes prepared by the method for preparing deer placental stem cell exosomes according to any one of claims 1-8.

10. The use of the deer placental stem cell exosomes of claim 9 in the preparation of tissue regeneration and repair agents, anti-inflammatory and anti-aging agents, skin care raw materials, targeted delivery carriers, and stem cell culture additives.

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