Ultrafiltration-size exclusion chromatography combined technology stem cell exosome extraction and freeze-drying preservation technology

Abstract

The invention provides an extraction and freeze-drying preservation technology of stem cell exosomes. The extraction and freeze-drying preservation technology comprises the following steps: (1) primarily concentrating the exosomes by adopting an ultrafiltration method; (2) further purifying by combining size exclusion chromatography (SEC), and removing impurity proteins and non-exosome vesicles; (3) a trehalose-nanogold composite protective agent is adopted for freeze-drying treatment, so that the structural stability of the exosome in the freeze-drying process is improved; and (4) optimizing a freeze-drying process, so that the exosome can be stably preserved for 12 months at room temperature. According to the method, the recovery rate and the biological activity of the exosome are remarkably improved, and the method is suitable for large-scale production and clinical application.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for efficient extraction and freeze-drying preservation of stem cell exosomes, particularly to ultrafiltration-size exclusion chromatography purification technology and a trehalose-nano gold protectant freeze-drying formulation, to improve the purity and stability of exosomes and extend their shelf life at room temperature. Background Technology

[0002] Exosomes are nanoscale vesicles (30-150 nm) secreted by cells, carrying bioactive substances such as proteins, nucleic acids, and lipids, and playing important roles in intercellular communication, immune regulation, and tissue repair. However, the extraction and preservation of exosomes still face the following challenges:

[0003] 1. Low extraction purity: Traditional ultracentrifugation methods are prone to contamination with protein aggregates or apoptotic bodies, affecting subsequent applications.

[0004] 2. Loss of activity: The freeze-thaw process can easily cause exosome membrane rupture and leakage of contents.

[0005] 3. Short shelf life: Liquid storage requires a low temperature of -80℃, resulting in high transportation and storage costs.

[0006] In existing technologies, ultrafiltration and size exclusion chromatography (SEC) have been used for exosome purification, but their use alone still suffers from low recovery rates or residual impurities. Furthermore, lyophilization protectants (such as trehalose) can reduce ice crystal damage, but their protective effect as a single component is limited. Therefore, there is an urgent need to develop an efficient and stable exosome extraction and lyophilization preservation technology. Summary of the Invention

[0007] The purpose of this invention is to provide a high-purity, high-yield method for exosome extraction and to develop a novel freeze-drying protectant to reduce freeze-drying damage to exosomes, effectively extend the shelf life of exosomes at room temperature to 12 months, and reduce storage and transportation costs. The following is a detailed explanation of the technical solution and its advantages.

[0008] 1. Exosome extraction technology

[0009] Ultrafiltration-size exclusion chromatography was employed.

[0010] Ultrafiltration step: Use a 100kDa ultrafiltration membrane to concentrate the culture supernatant and remove large particulate impurities.

[0011] SEC purification: Using a Sepharose CL-4B column, exosome peaks of 30-150 nm were collected to remove free proteins and extraneous vesicles.

[0012] 1.1 Preliminary Concentration via Ultrafiltration

[0013] 1.1.1 Sample Pretreatment

[0014] Collect the stem cell culture supernatant (recommended culture time 48-72 hours), centrifuge at 3000×g for 15 minutes (at 4℃), filter the supernatant through a 0.45μm filter membrane to remove residual cell debris, and proceed with subsequent processing immediately or temporarily store at 4℃ (not exceeding 6 hours).

[0015] 1.1.2 Preparation of Ultrafiltration System

[0016] A polyethersulfone (PES) ultrafiltration membrane with a molecular weight cutoff of 100 kDa was selected. Before use, the membrane assembly was rinsed with PBS buffer for 30 minutes. The system pressure was adjusted to 0.5-1.0 bar (adjusted according to the membrane area), and the circulation flow rate was set to 100-150 mL / min (tangential flow mode).

[0017] 1.1.3 Concentration Operation Procedure

[0018] Inject the pretreated sample into the ultrafiltration system at a constant flow rate, maintain the operating temperature at 4-10℃ (equipped with a cooling circulation system), monitor the transmembrane pressure (TMP) in real time and keep it below 1.5 bar. Stop the process when the sample volume is concentrated to 1 / 10-1 / 20 of the original volume, and perform two washes (diafiltration) with 1 / 10 of the original volume of PBS.

[0019] 1.1.4 Sample Recovery

[0020] Collect the retentate in a pre-chilled centrifuge tube and proceed immediately to the next purification step, or temporarily store at 4°C (for no more than 2 hours) and sterilize by filtration through a 0.22 μm filter membrane (if long-term storage is required).

[0021] 1.1.5 Quality Control

[0022] Sample and determine protein concentration (BCA method), measure sample conductivity (ensuring complete buffer replacement), and record the final concentration factor and recovery rate (typically >80%).

[0023] 1.1.6 Precautions

[0024] To prevent membrane fouling: Rinse with 0.1M NaOH for 30 minutes after each batch treatment to prevent foaming; add defoamer (such as Pluronic F-68) if necessary; regularly monitor membrane flux; replace the membrane module when the flux drops by more than 20%.

[0025] 1.2 Size Exclusion Chromatography (SEC) for Fine Purification

[0026] Use a Sepharose CL-4B or similar gel filtration column, with the column bed volume optimized based on the sample volume (typically 10-30 mL). Use PBS as the mobile phase, with a flow rate controlled at 0.5-1 mL / min to avoid shear damage to exosomes.

[0027] Collect the eluent and determine the exosome peaks (corresponding to the 30-150 nm fraction) by UV detection (280 nm) or nanoparticle tracking analysis (NTA). Combine the target peak fractions to avoid mixing in earlier protein peaks or later miscellaneous vesicle peaks.

[0028] 1.3 Verification of exosome purity

[0029] Nanoparticle tracking analysis (NTA): Detects particle size distribution, ensuring the main peak is within the 30-150 nm range. Use a commercial NTA instrument (such as the Malvern NanoSight NS300), and preheat the laser for at least 15 minutes (typically a 405 nm or 488 nm laser).

[0030] Instrument calibration: Calibration was performed using 100nm standard polystyrene microspheres to ensure that the detected particle size error was within ±5%. Appropriate camera parameters were set: Camera gain: 12-16, Detection threshold: 3-5, Shutter speed: 30ms.

[0031] Sample preparation: Dilute the exosome sample with PBS to a suitable concentration (10^7-10^8 particles / mL), vortex for 5 seconds after dilution to avoid bubble formation, and filter the sample through a 0.22 μm filter membrane to remove large particulate contaminants.

[0032] Detection procedure: Slowly inject 500 μL of sample into the sample cell using a 1 mL syringe. Set the detection temperature (usually 25°C) and detection time (60 seconds). Perform at least 3 independent measurements for each sample. Clean the sample cell 3 times with PBS before each measurement.

[0033] Data analysis: Use professional analysis software (such as NTA3.4 software) to process data.

[0034] Key parameters to record: particle size distribution histogram (main peaks should be between 30-150 nm), average particle size (Z-average), particle concentration (particles / mL), and exclusion of outliers (such as aggregates >300 nm).

[0035] Quality control: Verify instrument status with standards before each batch of testing. During sample testing, a PBS blank control must be run simultaneously. The relative standard deviation (RSD) of three repeated measurements should be <15%.

[0036] Precautions: Avoid excessive dilution or concentration of samples (affecting Brownian motion observation), avoid shaking the experimental table during the detection process, clean the sample cell regularly (using 1% Hellmanex solution), and save the original video file when storing data for a long time. Western Blot (WB): detect exosome marker proteins (such as CD63, TSG101) and exclude endoplasmic reticulum contamination (Calnexin negative).

[0037] Sample preparation: Exosome lysis, take 20 μg of exosomal protein sample, and add 4×Laemmli buffer (containing 2% SDS).

[0038] Proteins were denatured by heating in a 95℃ metal bath for 5 minutes. Protein concentration was determined using the BCA method. All samples were adjusted to the same concentration (1 μg / μL) with PBS. Electrophoresis separation gels were prepared: a 12% separating gel (lower layer) and a 5% stacking gel (upper layer). TEMED was added, and the gels were poured immediately to avoid air bubbles. For sample loading and electrophoresis, 20 μg of protein (volume ≤ 20 μL) was loaded into each well, along with pre-stained protein markers (10-180 kDa range). The initial voltage was 80V (stacking gel), adjusted to 120V after entering the separating gel. Electrophoresis was terminated when bromophenol blue reached the bottom of the gel. Wet transfer parameters were used: transfer buffer: 25 mM Tris, 192 mM glycine, 20% methanol; constant current 200 mA; transfer at 4℃ for 90 minutes using a 0.45 μm PVDF membrane (activated with methanol for 30 seconds). Transfer verification: Use Ponceau S staining to observe the transfer efficiency and check whether the marker bands are completely transferred.

[0039] Immunological testing: Block with 5% skim milk / TBST at room temperature for 1 hour, shaker speed: 60 rpm.

[0040] Primary antibody incubation: Antibody dilution ratio: CD63 (1:1000), TSG101 (1:800), Calnexin (1:1000), incubate overnight at 4°C (12-16 hours), wash membrane 3 times with TBST (10 minutes each time).

[0041] Secondary antibody incubation: HRP-labeled secondary antibody (1:5000 dilution) was incubated at room temperature for 1 hour, followed by TBST washing three times (10 minutes each time).

[0042] Colorimetric Development and Analysis: ECL colorimetric development was performed by mixing ECL chemiluminescence solutions A and B in a 1:1 ratio and uniformly adding the mixture to the membrane surface. Exposure was then performed using a chemiluminescence imaging system (30-60 seconds). Results interpretation: Positive criteria: CD63: band visible at 25-50 kDa; TSG101: band visible at 45 kDa; Calnexin: negative at 90 kDa. Grayscale analysis was performed using ImageJ software. Quality Control: For each batch of experiments, a positive control (known exosome sample) and a negative control (cell lysate) were set up.

[0043] To verify the consistency of loading amount for internal control proteins (such as β-actin), the experiment should be repeated ≥3 times.

[0044] Precautions: Wear gloves throughout the process to avoid membrane contamination. Be careful to remove air bubbles during membrane transfer. Use a fresh transfer membrane for different antibody detections. Store the primary antibody at 4°C (it can be reused 3-5 times). Transmission electron microscopy (TEM): Observe the vesicle morphology and confirm the typical cup-shaped structure.

[0045] 1.4 Technological Advantages

[0046] Compared to traditional ultracentrifugation, ultrafiltration-SEC coupling improves recovery by more than 30% and avoids vesicle rupture caused by high-speed centrifugation. Size exclusion chromatography effectively separates exosomes from non-vesicle proteins, reducing residual impurity proteins to <5%. The entire process is performed in a mild buffer system (PBS) to maximize the preservation of exosome biological activity.

[0047] 2. Freeze-drying protectant formulation

[0048] Trehalose-nano gold composite protective agent (mass ratio 5:1): Trehalose: reduces ice crystal formation and maintains membrane integrity.

[0049] Nano gold (5nm): Antioxidant, stabilizes exosome surface proteins.

[0050] 2.1 Composition and Function of Protective Agent

[0051] 2.1.1 Trehalose concentration range: 5-10% (w / v), preferably 8%. Mechanism of action: Through the "water substitution" hypothesis, it replaces water molecules in binding to the phospholipid membrane during freeze-drying, maintaining the integrity of the membrane structure. It inhibits ice crystal formation and reduces mechanical damage.

[0052] 2.1.2 Gold Nanoparticles (AuNPs)

[0053] Particle size: 5nm, surface modified with carboxylation (-COOH) to improve dispersibility.

[0054] Concentration range: 0.1-0.5 mg / mL, preferably 0.2 mg / mL.

[0055] Mechanism of action: Antioxidant: Neutralizes free radicals generated during freeze-drying, protecting exosome membrane lipids and proteins. Stabilizing surface proteins: Reduces protein aggregation through electrostatic interactions.

[0056] 2.1.3 Buffer System

[0057] Phosphate-buffered saline (PBS, pH 7.4) provides ion balance. 1 mM EDTA can be added to chelate metal ions and prevent oxidative damage.

[0058] 2.2 Preparation method of composite protective agent

[0059] Dissolve trehalose in PBS to a final concentration of 8% (w / v) and stir magnetically until completely dissolved. Add carboxylated gold nanoparticles (0.2 mg / mL) and sonicate (50 W, 5 min) to ensure uniform dispersion. Filter sterilize through a 0.22 μm membrane and store at 4°C for later use.

[0060] 2.3 Optimization and Validation of Protective Agents

[0061] Freeze-drying protection efficiency test: Experimental group: exosomes + trehalose-gold nanoparticle composite protectant. Control group: trehalose alone or gold nanoparticle protectant. Evaluation indicators: exosome recovery rate (BCA protein quantification), membrane integrity (dynamic light scattering detection of particle size change), and bioactivity (cellular uptake assay).

[0062] Long-term stability testing involved storing lyophilized exosomes at 4°C, 25°C, and 37°C, with periodic monitoring of activity retention. After 12 months of storage at 25°C, the exosome activity retention rate was >90% in the combined protectant group and <60% in the single protectant group.

[0063] 2.4 Technological Advantages

[0064] Trehalose and gold nanoparticles work synergistically to significantly reduce lyophilization damage compared to single protectants. The introduction of gold nanoparticles did not affect exosome function (e.g., cell targeting) and showed no cytotoxicity (MTT assay verification). The formulation has clearly defined components and meets clinical-grade formulation requirements (no animal-derived components, low endotoxin).

[0065] 3. Optimization of freeze-drying process

[0066] Pre-freeze: -80℃ for 2 hours, then transfer to a freeze dryer.

[0067] Primary drying: -40℃, 0.1mBar, 24h.

[0068] Secondary drying: 25℃, 0.01mBar, 12h.

[0069] 3.1 Pre-freezing stage

[0070] Sample pretreatment: The purified exosomes were mixed with a cryoprotectant (8% trehalose + 0.2 mg / mL gold nanoparticles) at a 1:1 volume ratio. The mixture was dispensed into sterile vials, with each vial containing no more than 1 / 3 of the vial volume (usually 2 mL). The pre-freezing procedure used a gradient cooling method: first, the temperature was lowered from room temperature to 4°C at a rate of 1°C / min and held for 30 minutes; then, the temperature was lowered from 4°C to -20°C at a rate of 1°C / min and held for 1 hour; finally, the temperature was lowered from -20°C to -80°C at a rate of 0.5°C / min and held in an ultra-low temperature freezer at -80°C for at least 2 hours to ensure complete freezing.

[0071] 3.2 Primary Drying Stage

[0072] Freeze dryer parameter settings: condenser temperature: -80℃, vacuum degree: 0.1mBar, baffle temperature: -40℃, duration: 24 hours. Process monitoring is performed by monitoring the sample resistance value using the resistance method. When the resistance is >1MΩ, it indicates that most of the free water has sublimated. The sample morphology is observed regularly to ensure that no collapse or melting occurs.

[0073] 3.3 Secondary drying stage parameter adjustment: Gradually increase the partition temperature to 25℃ (heating rate 0.2℃ / min), increase the vacuum to 0.01 mBar, duration: 12 hours. Endpoint determination: The temperature difference between the sample and the partition is <1℃, the vacuum stabilizes at 0.01 mBar for more than 2 hours, and the sample moisture content is <3% (Karl Fischer method).

[0074] 3.4 Post-processing and storage: After freeze-drying, the product is sealed by plugging under a dry nitrogen atmosphere and immediately transferred to a desiccator (relative humidity...).

[0075] <10%) Short- to medium-term storage. Preferred long-term storage conditions: temperature below 25℃, protected from light and moisture. Shelf life verification: activity retention >90% after 12 months.

[0076] 3.5 Process validation, physical property testing, reconstitution time: It should completely dissolve within 30 seconds after adding 1 mL of PBS; appearance:

[0077] It should be a white or pale yellow, loose, cake-like substance, without collapse or cracks. Bioactivity assay particle size distribution: DLS detection should be in the range of 30-150 nm; marker proteins: CD63 and TSG101 expression levels should be no less than 80% of those before lyophilization; functional verification: cellular uptake efficiency should be no less than 85% of that of the fresh sample.

[0078] 3.6 Technical advantages: The gradient pre-freezing process effectively avoids ice crystal damage, the two-stage drying ensures that the residual moisture content is <3%, the optimized heating rate avoids protein denaturation caused by phase transition, and the parameters of the whole process are controllable, making it suitable for large-scale production.

[0079] originality

[0080] 1. Technological combination innovation

[0081] 1.1 Optimization scheme for ultrafiltration-SEC coupled technology

[0082] 1.1.1 The "tangential flow ultrafiltration + precise molecular weight cutoff (100kDa)" pretreatment process is proposed for the first time.

[0083] 1.1.2 Innovative chromatography conditions using Sepharose CL-4B packing material (flow rate 0.5 cm / min, elution volume control) were employed.

[0084] 1.1.3 Establish a standardized recovery rate calculation formula: Recovery rate = (Number of particles after SEC / Number of particles before ultrafiltration) × 100%

[0085] 1.2 Breakthrough in the formulation of freeze-drying protectants

[0086] 1.2.1 A pioneering "trehalose-nano gold" synergistic protection system (mass ratio 5:1)

[0087] 1.2.2 Discovery of the dual mechanism of action of gold nanoparticles (5nm carboxylated) in exosome lyophilization:

[0088] Surface charge stabilizing effect (Zeta potential change < 2mV)

[0089] Membrane protein conformation protection (verified by circular dichroism chromatography)

[0090] 1.3 Creative improvements to the freeze-drying process

[0091] The criteria for determining the endpoint of freeze-drying were established for the first time.

[0092] Sample resistance > 1 MΩ

[0093] Temperature difference <1℃ for 2 hours

[0094] 2. Improved technical performance

[0095] 2.1 The extraction purity was significantly improved.

[0096] 2.1.1 Impurity protein removal rate >95% (40% improvement compared to ultracentrifugation) 2.1.2 Exosome recovery rate reaches 82.3±3.5% (approximately 50-60% with traditional methods) 2.2 Breakthrough in freeze-dried product stability

[0097] 2.2.1 The storage period at room temperature is extended from the usual 3 months to 12 months: the particle size change rate after 12 months is <5%.

[0098] Marker protein expression retention rate >90%

[0099] 2.2.2 Excellent reconstitution properties:

[0100] Reconstitution time <30 seconds

[0101] Aggregation rate after reconstitution <3%

[0102] 3. Enhanced application value

[0103] 3.1 Advantages of Clinical Translation

[0104] 3.1.1 Lyophilized formulations are easy to transport (can be transported at room temperature for 72 hours)

[0105] 3.1.2 Production costs are reduced by approximately 35% (compared to the liquid nitrogen preservation method).

[0106] 3.2 Feasibility of Standardized Production

[0107] 3.2.1 Establish a complete QC standard (including 6 key quality control indicators)

[0108] 3.2.2 Batch-to-batch variation RSD < 8% (meets GMP requirements)

[0109] 4. Biological effects guarantee

[0110] 4.1 Functional Integrity Verification

[0111] 4.1.1 Cell uptake efficiency remained at 92.3 ± 2.1% of that of fresh samples.

[0112] 4.1.2 Animal experiments showed equivalent therapeutic effects (wound healing model p>0.05)

[0113] 4.2 Security Confirmation

[0114] 4.2.1 No cytotoxicity (verified by CCK-8 assay)

[0115] 4.2.2 No pyrogenic reaction (negative rabbit pyrogen test) Attached Figure Description

[0116] Figure 1 Flowchart of stem cell exosome extraction and freeze-drying preservation technology.

[0117] Figure 2 Schematic diagram of an ultrafiltration-size exclusion chromatography coupled apparatus.

[0118] Figure 3 : Freeze-drying process flow diagram. Detailed Implementation

[0119] Example 1: Exosome Extraction and Purity Verification

[0120] 1.1 Experimental Materials

[0121] 1.1.1 Culture supernatant of human umbilical cord mesenchymal stem cells (hUC-MSCs) (collected after 72 hours)

[0122] 1.1.2 Main reagents:

[0123] 100kDa ultrafiltration centrifuge tubes (Millipore)

[0124] Sepharose CL-4B chromatography column (GE Healthcare)

[0125] 0.22μm PVDF filter membrane

[0126] 1.2 Experimental Procedure

[0127] 1.2.1 Ultrafiltration Concentration

[0128] 1.2.1.1 Centrifuge at 3000×g to remove cell debris.

[0129] 1.2.1.2 The supernatant was filtered through a 0.45 μm filter and then centrifuged at 4000 × g for 30 minutes using a 100 kDa ultrafiltration tube. 1.2.1.3 The supernatant was washed three times with PBS and finally concentrated to 1 / 20 of its original volume. 1.2.2 SEC purification.

[0130] 1.2.2.1 Equilibrate the chromatography column (1.6 × 60 cm) with PBS. 1.2.2.2 Loading volume ≤ 5% of column volume.

[0131] 1.2.2.3 Flow rate 0.5 mL / min, UV monitoring at 280 nm. 1.2.2.4 Collect tubes 8-12 (2 mL per tube).

[0132] 1.3 Results Analysis

[0133] 1.3.1 NTA Display:

[0134] Peak particle size: 98.3 ± 12.6 nm

[0135] Particle concentration: 3.2 × 10^10 particles / mL

[0136] 1.3.2 WB Detection:

[0137] CD63 / TSG101 strongly positive, Calnexin negative.

[0138] 1.3.3 Electron microscopy observation:

[0139] Typical cup-shaped vesicle structure

[0140] Protein aggregates

[0141] Example 2: Optimization of Lyophilization Protectant Formulation 2.1 Experimental Design

[0142] 2.1.1 Set up 4 comparison groups:

[0143] Group 1: 5% Trehalose

[0144] Group 2: 5% trehalose + 0.1 mg / mL gold nanoparticles; Group 3: 8% trehalose + 0.2 mg / mL gold nanoparticles; Group 4: 10% trehalose + 0.5 mg / mL gold nanoparticles. 2.2 Lyophilization Program

[0145] 2.2.1 Pre-freezing:

[0146] -80℃ gradient cooling (as described in 3.1.2)

[0147] 2.2.2 Freeze-drying:

[0148] Primary drying for 24 hours (-40℃)

[0149] Secondary drying for 12 hours (25℃)

[0150] 2.3 Performance Evaluation

[0151] 2.3.1 Reconstitution rate:

[0152] Group 3 was the best (98.2 ± 1.5%).

[0153] 2.3.2 Particle size stability:

[0154] Group 3: Changes before and after freeze-drying <5%

[0155] 2.3.3 Bioactivity:

[0156] Group 3 cells maintained 92.3% of the uptake efficiency of fresh samples.

[0157] Example 3: Long-term stability verification

[0158] 3.1 Storage conditions

[0159] 3.1.1 Temperature setting:

[0160] -80℃ (control), 4℃, 25℃, 37℃ (accelerated test) 3.1.2 Packaging:

[0161] Nitrogen protection in vials

[0162] Aluminum-plastic composite membrane seal

[0163] 3.2 Detection Time Point

[0164] 3.2.10 / 1 / 3 / 6 / 9 / 12 months

[0165] 3.3 Key Results

[0166] 3.3.125℃ storage:

[0167] 6 months: Activity retained at 95.6%

[0168] 12 months: Activity retention 89.2%

[0169] 3.3.237℃ accelerated test: 3 months is equivalent to 12 months at 25℃.

[0170] Example 4: Functional Verification

[0171] 4.1 Cell migration experiment

[0172] 4.1.1 Scratch test model:

[0173] Human dermal fibroblasts (HDF)

[0174] Scratch width 500μm

[0175] 4.1.2 Processing Group:

[0176] Fresh exosomes (50 μg / mL)

[0177] Lyophilized and reconstituted exosomes (50 μg / mL)

[0178] PBS control

[0179] 4.2 Results Analysis

[0180] 4.2.124-hour migration rate:

[0181] Fresh group: 78.3±5.2%

[0182] Freeze-dried group: 72.1 ± 4.8%

[0183] Control group: 21.5 ± 3.1%

[0184] 4.2.2 Statistical Analysis:

[0185] There was no significant difference between the freeze-dried group and the fresh group (p>0.05). Example 5: Scale-up production verification 5.1 Scale-up parameters

[0186] 5.1.1 Ultrafiltration scale:

[0187] Tangential flow system (membrane area 0.1m²) 2 )

[0188] Processing capacity 5L / batch

[0189] 5.1.2 SEC purification:

[0190] Industrial-grade chromatography column (10cm diameter)

[0191] Linear scaling up while maintaining the same flow rate (0.5 cm / min)

[0192] 5.2 Quality Control Data

[0193] 5.2.1 Batch-to-batch differences:

[0194] Particle size RSD < 8%

[0195] Protein recovery rate RSD < 10%

[0196] 5.2.2 Sterility test:

[0197] All batches meet pharmacopoeia standards.

[0198] This invention improves the purity of exosomes through ultrafiltration-SEC combined technology, and, combined with trehalose-nano gold freeze-drying protectant, significantly enhances the freeze-drying stability and room temperature storage period of exosomes, providing a feasible solution for the large-scale production and clinical application of exosomes.

Claims

1. A method for extracting stem cell exosomes, characterized in that... Includes the following steps: The stem cell culture supernatant was concentrated by tangential flow filtration using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. Size exclusion chromatography was performed using a Sepharose CL-4B column, with the elution flow rate controlled at 0.5-1 mL / min. Collect elution fractions with particle sizes ranging from 30 to 150 nm.

2. The extraction method according to claim 1, characterized in that: The ultrafiltration process is carried out at 4-10°C with a transmembrane pressure ≤1.5 bar; The size exclusion chromatography used PBS as the mobile phase.

3. An exosome lyophilization protectant, characterized in that... It consists of the following components: Trehalose: 5-10% (w / v); Carboxylated gold nanoparticles (5nm): 0.1-0.5mg / mL; The remainder is PBS buffer (pH 7.4).

4. The freeze-drying protectant according to claim 3, characterized in that: The mass ratio of trehalose to nano-gold is 5:1; The degree of carboxylation modification on the surface of the gold nanoparticles is ≥80%.

5. A method for preparing an exosome lyophilized formulation, characterized in that... include: The exosomes extracted according to claim 1 are mixed with the protective agent according to claim 3 at a volume ratio of 1:1; Gradient pre-freezing: cooling in stages from 4℃ to -20℃ to -80℃; Primary drying: Drying at -40℃ and 0.1 mBar for 24 hours; Secondary drying: Drying at 25℃ and 0.01mBar for 12 hours.

6. The preparation method according to claim 5, characterized in that: The criterion for determining the end point of freeze-drying is that the sample resistance is >1MΩ and the temperature difference is <1℃ for 2 hours. The moisture content after freeze-drying is ≤3%.

7. A lyophilized exosome preparation prepared by the method of claim 5, characterized in that: The activity retention rate is ≥90% after 12 months of storage at room temperature; Reconstitution time ≤ 30 seconds; Particles with a size distribution of 30-150 nm account for ≥95%.

8. The use of the lyophilized exosome preparation according to claim 7 in the preparation of drugs for treating tissue damage.

9. A quality control standard for exosomes, characterized in that... include: NTA testing: ≥90% of particles are 30-150nm; Western Blot analysis: CD63 / TSG101 positive and Calnexin negative; The aseptic testing of the lyophilized preparations meets the standards of the Chinese Pharmacopoeia.

10. An exosome production system, characterized in that... include: Tangential flow ultrafiltration unit (equipped with a 100kDa membrane); Size exclusion chromatography purification module; Programmable freeze dryer (including gradient cooling function).

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