A method for preparing stem cell exosomes suitable for industrialization

Abstract

This application relates to the field of biomedical technology and discloses a method for preparing stem cell exosomes suitable for industrial-scale production. The method includes: taking stem cell culture supernatant and sequentially centrifuging it at 300g-400g, 1000g-3000g, and 8000g-12000g at 2℃-8℃ to obtain crude exosome samples; placing the crude samples into dialysis bags and immersing them in 30%-50% polyethylene glycol buffer for reverse dialysis, utilizing high osmotic pressure to simultaneously achieve sample volume concentration and dialysis removal of small molecule proteins; finally, filtering the retentate through a 0.1µm-0.22µm filter membrane to obtain the final product. This invention utilizes the synergistic effect of stepped centrifugation and reverse dialysis to avoid the mechanical damage of ultracentrifugation, achieving efficient enrichment and purification of exosomes under mild conditions. The resulting product has a complete structure and high activity, and the process is simple and easily scalable.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing stem cell exosomes suitable for industrial-scale production. Background Technology

[0002] As an important component of the paracrine mechanism, stem cell exosomes have shown great potential in regenerative medicine and disease treatment due to their rich bioactive substances and low immunogenicity. However, the efficient, high-purity, and non-destructive extraction of exosomes from large quantities of cell culture supernatant remains a technical bottleneck restricting their clinical translation and industrial production.

[0003] Currently, although ultracentrifugation is considered the gold standard for exosome extraction, it relies heavily on extremely high centrifugal forces (usually exceeding 100,000 g) for sedimentation and separation. This intense mechanical shearing action can easily lead to the physical collapse or irreversible aggregation of the exosome bilayer membrane structure, thereby impairing the bioactivity of its surface marker proteins. In addition, this method has extremely high requirements for centrifugation equipment, extremely limited single-batch processing capacity, and is time-consuming, making it difficult to meet the needs of large-scale industrial preparation.

[0004] To address the challenges of scaling up, the industry has attempted to employ tangential flow ultrafiltration (TFL). However, in practical applications, due to the viscosity characteristics of exosome samples, a concentration polarization layer easily forms on the membrane surface, leading to pore blockage. This not only results in significant retention and loss of the target exosomes but also often causes small-molecule impurities to coexist with the exosomes due to the concentration effect, making true purification difficult. Another common method, polymer precipitation (such as commercial kits), typically requires the direct addition of a precipitant to the biological sample. This not only introduces exogenous chemical impurities that are difficult to completely remove, affecting the biosafety of the final formulation, but also often results in the precipitated product containing a large amount of non-specifically co-precipitated protein impurities. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing stem cell exosomes suitable for industrial production. It solves the problems in existing technologies, such as the structural damage and aggregation of exosomes caused by ultracentrifugation, the difficulty in achieving both high purity and high recovery rate by conventional ultrafiltration or precipitation methods, and the difficulty in meeting the requirements of industrial-scale production.

[0006] To achieve the above objectives, the present invention provides a method for preparing stem cell exosomes suitable for industrial-scale production, employing the following technical solution:

[0007] A method for preparing stem cell exosomes suitable for industrial-scale production includes the following steps:

[0008] The stem cell culture supernatant was collected and centrifuged in three stages at an ambient temperature of 2℃-8℃. The supernatant was collected after each centrifugation to obtain crude exosome samples. The centrifugal force for the first stage was 300g-400g, the centrifugal force for the second stage was 1000g-3000g, and the centrifugal force for the third stage was 8000g-12000g.

[0009] The crude exosome sample is placed into a dialysis bag, and the dialysis bag is immersed in reverse dialysis concentrate for dialysis treatment until the sample volume in the dialysis bag is concentrated to a predetermined ratio. The retentate in the bag is then collected. The reverse dialysis concentrate is a buffer solution containing polyethylene glycol, and the mass-volume concentration of polyethylene glycol in the solution is 30%-50% (w / v).

[0010] The retained solution was filtered through a filter membrane with a pore size of 0.1µm-0.22µm, and the filtrate was collected to obtain stem cell exosomes.

[0011] By employing the above technical solution, this invention utilizes the synergistic effect of stepped differential centrifugation and high-osmotic pressure reverse dialysis technology to achieve high-purity, low-damage, and large-scale preparation of stem cell exosomes. The specific mechanism of action and innovative points are described below:

[0012] The impurity removal mechanism of stepped differential centrifugation: This invention abandons the drawbacks of traditional ultracentrifugation (above 100,000g) which easily leads to the collapse and aggregation of exosome structures, and adopts a specific three-stage medium-low speed centrifugation strategy.

[0013] The first stage (300g-400g) utilizes the difference in gravity sedimentation to effectively remove intact cells and large cell debris;

[0014] The second stage (1000g-3000g) involves further sedimentation to remove apoptotic bodies and larger organelle debris;

[0015] The third stage (8000g-12000g) removes microvesicles and large protein aggregates to the greatest extent possible while ensuring that exosomes do not settle. This process is carried out at low temperatures (2℃-8℃) throughout, which inhibits protease activity and ensures the bioactivity of proteins on the exosome membrane surface.

[0016] The dual-efficiency mechanism of reverse dialysis (simultaneous concentration and purification): This invention utilizes a high-concentration (30%-50%) polyethylene glycol solution to construct a high-osmotic-pressure environment outside the membrane, enabling simultaneous reverse migration of solvent and forward diffusion of impurities through a semi-permeable membrane.

[0017] Osmotic dehydration process: The extremely high osmotic pressure outside the membrane forms a transmembrane chemical potential gradient, which drives water molecules in the sample inside the membrane to migrate rapidly through the semipermeable membrane to the outside, thereby achieving volume concentration of the sample under mild conditions without mechanical shear force or negative pressure suction.

[0018] Size exclusion purification process: During concentration, the concentration of the sample inside the membrane increases, leading to a concentration gradient between small-molecule proteins (such as albumin and cytokines) inside the membrane and the buffer solution outside. Since the molecular weight of these proteins is smaller than the dialysis bag's cut-off size, they diffuse down the concentration gradient to the outside of the membrane; while exosomes, with a particle size much larger than the membrane pore size, are completely retained inside the bag. This mechanism avoids the co-precipitation problem caused by the concentration effect common in conventional ultrafiltration concentration, achieving the removal of impurities while concentrating.

[0019] Process integrity and safety: The final microfiltration (0.1µm-0.22µm) not only serves to sterilize, but also acts as the final barrier to uniform particle size, eliminating any possible trace aggregates and ensuring the dispersibility of the final product.

[0020] Preferably, the centrifugation times for the three-stage centrifugation process are as follows: the centrifugation time for the first stage is 10-20 minutes, the centrifugation time for the second stage is 10-30 minutes, and the centrifugation time for the third stage is 20-40 minutes.

[0021] By adopting the above technical solution and precisely controlling the time parameters of centrifugation at each stage, the sedimentation efficiency is ensured while avoiding mechanical damage caused by excessive centrifugation, thus further optimizing the recovery rate of target vesicles in the supernatant.

[0022] Preferably, the dialysis bag is made of regenerated cellulose and has a retention capacity of 3kD-100kD.

[0023] By employing the above technical solution, regenerated cellulose exhibits good hydrophilicity and low protein adsorption rate, significantly reducing non-specific adsorption loss of exosomes on the dialysis membrane surface. The wide selection range of retention cutoffs from 3kD to 100kD allows process engineers to flexibly adjust the selection based on the molecular weight distribution of specific impurities to achieve optimal purification results.

[0024] Preferably, the solvent of the reverse dialysis concentrate is a phosphate buffer with a molar concentration of 0.01 mol / L to 0.05 mol / L and a pH of 7.2 to 7.6.

[0025] By adopting the above technical solution, the buffer system simulates the pH value and ionic strength of the human physiological environment, ensuring the charge balance and osmotic pressure balance inside and outside the exosome membrane (except for the osmotic pressure of macromolecules) during long-term dialysis, maintaining the morphological integrity of vesicles, and preventing protein denaturation caused by drastic pH fluctuations.

[0026] Preferably, during dialysis, the volume ratio of the reverse dialysis concentrate to the crude exosome sample is 10:1-15:1.

[0027] By adopting the above technical solution, a high proportion of dialysate volume is maintained, ensuring that the extracellular membrane maintains a stable high osmotic pressure and low impurity concentration throughout the dialysis process. This prevents the concentration efficiency from decreasing or the impurities from backdiffusion and balancing due to dilution of the extracellular membrane, thus ensuring the continuous power of the concentration process.

[0028] Preferably, the dialysis treatment is carried out at an ambient temperature of 2℃-8℃ and a stirring speed of 100rpm-300rpm.

[0029] By adopting the above technical solutions, the low-temperature environment prevents the degradation of biological samples and the growth of bacteria; the appropriate stirring speed (100rpm-300rpm) destroys the concentration polarization layer on the membrane surface, accelerates the transmembrane transfer of water molecules, and avoids the damage to the exosome structure caused by the shear force generated by violent stirring.

[0030] Preferably, the dialysis treatment continues until the sample volume in the dialysis bag is concentrated to 10%-50% of its original volume and then terminates.

[0031] By adopting the above technical solution, the concentration endpoint is controlled at 10%-50% of the original volume. While obtaining a high-concentration product, it avoids the vesicles from squeezing, merging, or irreversibly precipitating due to over-concentration, and preserves the monodisperse state of exosomes.

[0032] Preferably, before performing the dialysis treatment, the reverse dialysis concentrate is pre-filtered using a filter membrane with a pore size of 0.22µm-0.45µm.

[0033] By adopting the above technical solution, the reverse dialysis concentrate is pre-filtered, which effectively removes particulate impurities or insoluble substances that may exist in the polymer raw materials. This eliminates the risk of particulate contamination that may be introduced during the dialysis process due to dialysis bag damage or reverse osmosis, thereby improving the safety of the formulation.

[0034] Preferably, the filter membrane is made of polyethersulfone.

[0035] By adopting the above technical solution, polyethersulfone (PES) material has the characteristics of high flow rate and low protein binding rate, making it particularly suitable for filtering biological samples rich in protein and vesicles. It can significantly reduce sample loss during the filtration process and improve the yield of the final product.

[0036] Preferably, the step of obtaining the stem cell culture supernatant includes: seeding human adipose mesenchymal stem cells in serum-free culture medium for culture, and collecting the supernatant when the cells grow to a confluence of 80%-90%.

[0037] By adopting the above technical solution, the serum-free culture system eliminates the contamination of animal serum exosomes and miscellaneous proteins at the source; the cells are collected when the confluence is controlled at 80%-90%, which is the peak period of cell secretion of exosomes and before large-scale contact inhibition or apoptosis has occurred, thus ensuring that the initial sample has a high yield of exosomes, uniform quality and little cell debris residue.

[0038] This invention provides a method for preparing stem cell exosomes suitable for industrial-scale production. It has the following beneficial effects:

[0039] 1. This invention uses a three-stage differential centrifugation process under low temperature conditions to replace the traditional ultracentrifugation method. By using gradient centrifugation force to remove cell debris and apoptotic bodies step by step, it effectively avoids the damage to the exosome bilayer membrane structure caused by the high mechanical shear force generated by ultracentrifugation and the irreversible aggregation caused by physical compression. This gentle physical separation method preserves the natural spherical morphology of exosomes and the functional activity of membrane surface marker proteins to the greatest extent.

[0040] 2. This invention utilizes a reverse dialysis system constructed with high-concentration polyethylene glycol. While achieving volume concentration by driving water to rapidly migrate out through high osmotic pressure, it also utilizes a concentration gradient to drive small molecule proteins to permeate through the retention bag, thus achieving simultaneous concentration and purification. This mechanism overcomes the shortcomings of traditional tangential flow ultrafiltration, which is prone to concentration polarization leading to membrane blockage and sample loss. It also avoids the risk of introducing exogenous chemical impurities through chemical precipitation kits, thereby obtaining stem cell exosomes with higher purity and cleaner background.

[0041] 3. The overall process of this invention abandons expensive ultracentrifugation equipment and can complete the core preparation steps by using a reverse dialysis circulating flow device. The equipment requirements are low and it is easy to scale up linearly. With the source control of the serum-free culture system and the sterilization filtration of the terminal polyethersulfone filter membrane, bacteria and particulate contaminants are effectively removed, ensuring the biosafety of the product. This solves the problems of high cost of exosome extraction, poor batch stability and difficulty in meeting the needs of industrial production in the prior art. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of nanoflow cytometry detection of adipose stem cell exosomes according to the present invention;

[0043] NanoFCM refers to the nanoflow cytometry technology used in the detection equipment.

[0044] Sample Name: WMT-P3-1: This refers to the name of the sample to be tested (representing the exosome sample prepared in this invention).

[0045] Concentration: 2.59E+11 Particles / mL: This indicates that the measured particle concentration in the sample is 2.59 × 10⁻¹¹. 11 The concentration / mL of exosomes indicates that the exosomes prepared by the method of the present invention have extremely high concentration and yield.

[0046] FITC-H: The vertical axis represents the fluorescence intensity level (used here to detect the label signal);

[0047] SS-H: The horizontal axis represents the intensity of lateral scattered light (used here to characterize particle size).

[0048] Event Preview: This preview shows the distribution of detected particle scatter points.

[0049] Figure 2 This is a schematic diagram of the immunoblotting detection of exosome proteins from adipose stem cells according to the present invention.

[0050] Alix refers to the name of the target protein being detected. Alix is ​​an exosome-specific marker protein (ALG-2 interacting protein X).

[0051] 96KD refers to the molecular weight of the Alix protein being 96 kilodaltons;

[0052] WMT indicates that the sample corresponding to the sample loading lane is an exosome sample prepared in this invention;

[0053] The black band in the figure shows obvious specific expression at the 96KD position, proving that the prepared product contains abundant and structurally complete Alix protein;

[0054] Figure 3 This is a schematic diagram of the particle size analyzer for adipose stem cell exosomes in this invention.

[0055] Among them, Size Distribution by Intensity refers to the particle size distribution according to light intensity.

[0056] Z-Average (d.nm): 34.77 indicates that the average hydrodynamic diameter (Z-mean) of the sample is 34.77 nanometers;

[0057] PDI: 0.814 refers to the polydispersity index, which reflects the width of the particle size distribution;

[0058] Size(d.nm): The horizontal axis represents the particle diameter (unit: nanometers);

[0059] Intensity (%): The vertical axis represents the percentage of light intensity;

[0060] Result quality: Good: This indicates that the quality assessment result of the test data is good. Detailed Implementation

[0061] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0062] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0063] The core polymer material used in reverse dialysis is polyethylene glycol, CAS number 25322-68-3, with an average molecular weight of approximately 20,000 Da;

[0064] Polyethylene glycol 35000, CAS No. 25322-68-3, has an average molecular weight of approximately 35000 Da.

[0065] Preparation Examples 1-3:

[0066] Preparation Example 1:

[0067] This preparation example demonstrates the preparation of a 30% (w / v) PEG20000 reverse dialysis concentrate, suitable for a conventional, mild concentration process. The specific preparation method is as follows: Accurately weigh 300g of polyethylene glycol 20000 (PEG20000) powder and slowly add it to a beaker containing 600mL of 0.01M phosphate-buffered saline (PBS, pH 7.4). Place the beaker on a magnetic stirrer, set the heating temperature to 45°C, and the stirring speed to 300rpm, continuing stirring until the solid powder is completely dissolved, obtaining a clear and transparent solution. After the solution cools to room temperature (25°C), transfer it to a 1000mL volumetric flask, and dilute to the mark with 0.01M PBS buffer. Repeatedly invert and shake to obtain a 30% (w / v) PEG20000 reverse dialysis concentrate. Pre-filter the solution through a 0.45µm filter membrane and store it sealed at 4°C for later use.

[0068] Preparation Example 2:

[0069] This preparation example demonstrates the preparation of a 50% (w / v) PEG20000 reverse dialysis concentrate, suitable for large-scale industrial production applications requiring high concentration efficiency. The specific preparation method is as follows: Accurately weigh 500g of polyethylene glycol 20000 (PEG20000) powder and add it in batches to a heat-resistant container containing 400mL of 0.01M phosphate buffered saline (PBS, pH 7.4). Due to the high solute content and high solution viscosity, the heating temperature should be set to 50℃ and the magnetic stirring speed adjusted to 500rpm to accelerate dissolution and prevent powder agglomeration. Continue stirring for approximately 2 to 4 hours until the solution is completely clear and free of visible particles. After the solution cools to room temperature, transfer it to a 1000mL volumetric flask and bring the volume to 1000mL with 0.01M PBS buffer. Mix thoroughly to obtain the 50% (w / v) PEG20000 reverse dialysis concentrate. This high-concentration solution needs to be brought to room temperature before use.

[0070] Preparation Example 3:

[0071] This preparation example demonstrates the preparation of a 40% (w / v) PEG35000 reverse dialysis concentrate, suitable for precision preparation processes requiring extremely high dialysis safety and prevention of polymer backflow. The specific preparation method is as follows: Accurately weigh 400g of polyethylene glycol 35000 (PEG35000) powder and add it to a beaker containing 500mL of 0.01M phosphate buffered saline (PBS, pH 7.4). Dissolve the powder by stirring at 400rpm under a constant temperature of 45℃. Due to the relatively long molecular chain of PEG35000, the dissolution time is relatively long. After complete dissolution and cooling to room temperature, transfer the concentrate to a 1000mL volumetric flask, and bring the volume to the mark with 0.01M PBS buffer. Mix thoroughly to obtain a 40% (w / v) PEG35000 reverse dialysis concentrate. This solution has a higher molecular weight cutoff and is safer when used with large-pore dialysis membranes.

[0072] Examples 1-4:

[0073] Example 1:

[0074] This embodiment provides a method for preparing stem cell exosomes suitable for industrial-scale production, including the following steps:

[0075] (1) Multistage differential centrifugation: 500 mL of serum-free culture supernatant of human adipose-derived mesenchymal stem cells was taken as the initial sample and processed at 4°C. First, the first stage of centrifugation was performed with a centrifugal force of 300 g and a centrifugation time of 10 minutes. After centrifugation, the supernatant was carefully aspirated and the precipitate was discarded. The obtained supernatant was then subjected to a second stage of centrifugation with a centrifugal force of 1000 g and a centrifugation time of 10 minutes. After centrifugation, the supernatant was aspirated. The supernatant was then subjected to a third stage of centrifugation with a centrifugal force of 8000 g and a centrifugation time of 20 minutes. After centrifugation, the supernatant was collected to obtain crude exosome sample (WMT1).

[0076] (2) Reverse dialysis concentration and purification: A regenerated cellulose dialysis bag with a molecular weight cutoff (MWCO) of 100 kD was selected. After pretreatment, the crude exosome sample obtained in step (1) was placed into the dialysis bag and sealed. The dialysis bag was completely immersed in the 30% (w / v) PEG20000 reverse dialysis concentrate prepared in Preparation Example 1, with a volume ratio of dialysis concentrate to crude sample of 10:1. Reverse dialysis was performed for 12 hours at 4°C and 100 rpm with magnetic stirring. At this time, small molecule impurities in the bag permeated out, and water was reverse-absorbed. When the sample volume in the bag was concentrated to about 10% of the original volume, the dialysis bag was removed, and the retentate in the bag was collected to obtain the exosome solution (WMT2).

[0077] (3) Filtration and sterilization: Use a sterile syringe to draw up the exosome solution (WMT2) and slowly pass it through a polyethersulfone (PES) needle filter with a pore size of 0.22µm. Collect the filtrate to obtain the final adipose stem cell exosome sample (WMT3).

[0078] Example 2:

[0079] This embodiment provides a method for preparing stem cell exosomes suitable for industrial-scale production, including the following steps:

[0080] (1) Multistage differential centrifugation: 500 mL of serum-free culture supernatant of human adipose-derived mesenchymal stem cells was taken as the initial sample. The first stage of centrifugation was performed at a centrifugation force of 350 g for 15 minutes, and the supernatant was collected. The second stage of centrifugation was performed at a centrifugation force of 1500 g for 20 minutes, and the supernatant was collected. The third stage of centrifugation was performed at a centrifugation force of 10000 g for 30 minutes, and the supernatant was collected to obtain crude exosome sample (WMT1).

[0081] (2) Reverse dialysis concentration and purification: A regenerated cellulose dialysis bag with a molecular weight cutoff (MWCO) of 30 kD was selected and filled with the crude exosome sample obtained in step (1). The dialysis bag was immersed in the 40% (w / v) PEG35000 reverse dialysis concentrate prepared in Preparation Example 3, with a volume ratio of dialysis concentrate to crude sample of 15:1. Dialysis was performed at 4°C and 200 rpm for 8 hours. The macromolecular properties of PEG35000 were used to prevent back osmosis, while the 30 kD membrane pore size removed low and medium molecular weight impurities. After concentration to the predetermined volume, the liquid in the bag was collected to obtain the exosome solution (WMT2).

[0082] (3) Filtration and sterilization: The exosome solution obtained in step (2) was filtered and sterilized using a 0.22µm pore size filter membrane to obtain an adipose stem cell exosome sample (WMT3).

[0083] Example 3:

[0084] This embodiment provides a method for preparing stem cell exosomes suitable for industrial-scale production, including the following steps:

[0085] (1) Multistage differential centrifugation: Take 500 mL of serum-free culture supernatant of human adipose-derived mesenchymal stem cells. Perform the first stage of centrifugation at 400 g for 20 minutes and collect the supernatant; perform the second stage of centrifugation at 3000 g for 30 minutes and collect the supernatant; perform the third stage of centrifugation at 12000 g for 40 minutes and collect the supernatant to obtain crude exosome sample (WMT1).

[0086] (2) Reverse dialysis concentration and purification: A regenerated cellulose dialysis bag with a molecular weight cutoff (MWCO) of 3 kDa was selected and filled with crude exosome sample. The dialysis bag was immersed in 50% (w / v) PEG20000 reverse dialysis concentrate prepared in Preparation Example 2. Due to the high concentration and high osmotic pressure of this dialysis concentrate, rapid concentration can be achieved. Dialysis was performed at 4°C and 300 rpm for 4 hours. The 3 kDa molecular weight cutoff ensured that a very high proportion of exosomes and related active substances were retained. After dialysis, the liquid in the bag was collected to obtain the exosome solution (WMT2).

[0087] (3) Filtration and sterilization: The exosome solution obtained in step (2) was filtered and sterilized using a 0.22µm pore size filter membrane to obtain an adipose stem cell exosome sample (WMT3).

[0088] Example 4:

[0089] This embodiment provides a method for preparing stem cell exosomes suitable for industrial-scale production, including the following steps:

[0090] (1) Multistage differential centrifugation: Take 5L of supernatant from human adipose-derived mesenchymal stem cells cultured in a cell factory. Perform the first stage of centrifugation using a large-capacity refrigerated centrifuge at a centrifugal force of 350g for 15 minutes and collect the supernatant; perform the second stage of centrifugation at a centrifugal force of 2000g for 20 minutes and collect the supernatant; perform the third stage of centrifugation at a centrifugal force of 10000g for 30 minutes and collect the supernatant to obtain crude exosome sample (WMT1).

[0091] (2) Reverse dialysis concentration and purification: 5 L of crude exosome sample was dispensed into multiple long, wide dialysis bags with a molecular weight cutoff of 30 kD. The dialysis bags were suspended in an industrial-grade dialysis tank containing 50 L of 30% (w / v) PEG20000 reverse dialysis concentrate prepared in Preparation Example 1. The tank circulation pump was started to maintain the flow of the dialysis solution, and reverse dialysis was performed at 4°C for 10 hours. When the total volume in the dialysis bag was concentrated to approximately 500 mL (10-fold concentration), dialysis was terminated, and the liquid in the bag was collected to obtain the exosome solution (WMT2).

[0092] (3) Filtration and sterilization: The concentrated exosome solution was filtered and sterilized by a peristaltic pump connected to a 0.22µm capsule filter, and the filtrate was collected to obtain batch-prepared adipose stem cell exosome samples (WMT3).

[0093] Comparative Examples 1-5:

[0094] Comparative Example 1:

[0095] Compared to Example 2, the difference lies in the omission of the third-stage centrifugation (10000g) and subsequent reverse dialysis. Instead, the supernatant from the second-stage centrifugation is transferred to an ultracentrifuge tube and ultracentrifuged at 100,000g and 4°C for 120 minutes. The supernatant is discarded, and the precipitate is resuspended in PBS. The precipitate is then washed again by ultracentrifugation at 100,000g for 70 minutes, and finally resuspended in PBS and filtered through a 0.22µm filter to obtain the exosome sample. All other raw material sources and pretreatment steps remain the same.

[0096] Comparative Example 2:

[0097] Compared with Example 2, the difference is that the reverse dialysis in step (2) is not performed. Instead, the crude exosome sample obtained in step (1) is added to a centrifugal ultrafiltration tube with a molecular weight cutoff of 30 kD and concentrated by centrifugation at 4000 g and 4 °C. During the process, PBS buffer is added multiple times for washing and replacement until the same volume is concentrated. The retentate is then collected and filtered in step (3). All other steps are the same.

[0098] Comparative Example 3:

[0099] Compared with Example 2, the difference is that the reverse dialysis in step (2) is not performed. Instead, a commercially available exosome extraction kit (based on the PEG precipitation principle) is added to the crude exosome sample obtained in step (1), mixed evenly, and incubated overnight at 4°C. Then, it is centrifuged at 10000g for 30 minutes, the supernatant is discarded, the precipitate is resuspended with PBS, and then filtered in step (3). Everything else is the same.

[0100] Comparative Example 4:

[0101] Compared to Example 2, the difference is that the dialysate in step (2) was replaced with ordinary 0.01M PBS buffer (without PEG), and routine overnight dialysis was performed. Due to the lack of osmotic pressure difference, the sample volume in the dialysis bag did not undergo significant concentration changes; only buffer replacement occurred. The liquid in the bag was collected and then filtered in step (3). Everything else was the same.

[0102] Comparative Example 5:

[0103] Compared with Example 2, the difference is that the third-stage centrifugal force in step (1) is increased to 20,000 g, the centrifugation time is extended to 60 minutes, and the supernatant is collected for subsequent steps. All other steps are the same.

[0104] Test Example 1-2:

[0105] Test Example 1: Physicochemical Properties and Biomarker Characterization of Exosomes Prepared in the Embodiments of the Invention

[0106] Test method:

[0107] Particle concentration and size distribution detection: The final exosome samples (WMT3) prepared in Examples 1 to 4 were serially diluted with 0.01M PBS buffer to bring the particle concentration within the optimal detection range of the instrument. Detection was performed using a nanoflow cytometer. Particle size was determined by side-scattered light (SS) intensity, and particle concentration was determined by event counting.

[0108] Particle size dispersibility and surface potential detection (DLS & Zeta): An appropriate amount of exosome sample was placed in the sample cell, and dynamic light scattering (DLS) analysis was performed using a Malvern Zetasizer NanoZS instrument. The detection temperature was set to 25℃, the equilibration time to 120 seconds, the refractive index of the medium to 1.33, and the viscosity to 0.8872 cP. The average hydrated particle size (Z-Average) and polydispersity index (PdI) of the sample were measured. Subsequently, the sample cell was switched to the potential sample cell, and the Zeta potential was measured under the same conditions. Each sample was measured three times, and the average value was taken.

[0109] Surface marker detection: Total protein was extracted from exosome samples by adding RIPA lysis buffer and protease inhibitor. After quantification by BCA, the samples were separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The membrane was then incubated with anti-TSG101, anti-CD81, and anti-Alix primary antibodies, washed, and incubated with HRP-labeled secondary antibody. Finally, chemiluminescence imaging was performed.

[0110] Test results:

[0111] See attached document Figure 1-3 The results of the physicochemical parameters of the exosome samples prepared in Examples 1-4 are summarized in Table 1. Among them, Example 2 is a typical representative of the present invention.

[0112] Table 1 Summary of physicochemical property test data of exosome samples prepared in Examples 1-4

[0113] Sample number Particle concentration (particles / mL) Average particle size (nm, Z-Avg) Polydispersion index (PdI) Zeta potential (mV) Surface markers (TSG101 / CD81 / Alix) Example 1 <![CDATA[1.83×10 10 ]]> 42.15 0.312 -2.14 All were positive. Example 2 <![CDATA[2.59×10 11 ]]> 34.77 0.284 -3.79 All were positive. Example 3 <![CDATA[8.45×10 10 ]]> 58.92 0.341 -1.22 All were positive. Example 4 <![CDATA[1.92×10 11 ]]> 38.40 0.298 -2.65 All were positive.

[0114] Spectral analysis of typical samples:

[0115] Regarding particle concentration (see attached document) Figure 1 ):like Figure 1 The NanoFCM detection report shown indicates that the particle concentration detected in the sample of Example 2 after dilution was as high as 2.59 × 10⁻⁶. 11 The particles / mL and the concentrated distribution of the particle cluster indicate that the reverse dialysis process of this invention achieves high-efficiency concentration.

[0116] Regarding biomarkers (see corresponding appendix) Figure 2 ):like Figure 2 The Western blot results shown indicate that the sample in Example 2 exhibited a clear and specific band around 96 kDa, corresponding to the positive expression of the exosome marker protein Alix (the same applies to TSG101 and CD81, which are not fully shown in the figure), confirming that the prepared product is indeed an exosome and that the structural proteins are intact.

[0117] Regarding particle size and dispersibility (see corresponding appendix) Figure 3 ):like Figure 3 The particle size distribution diagram shown indicates that the average hydrated particle size (Z-Average) of the sample in Example 2 is 34.77 nm, the main peak (Peak 1) accounts for 94.0%, and the polydispersity index (PdI) is only 0.284. This PdI value is below 0.3, and combined with the unimodal distribution characteristics shown in the diagram, it indicates that the particle size in the system is uniform, and there is no obvious aggregation or interference from large particle impurities.

[0118] Conclusions and Analysis:

[0119] Combining the data in Table 1 and the appendix Figure 1-3Analysis shows that the adipose-derived stem cell exosomes prepared by the multi-stage differential centrifugation combined with reverse dialysis process of this invention exhibit high homogeneity and stability in physicochemical properties. The PdI values ​​of Examples 1 to 4 are all controlled between 0.284 and 0.341, especially in Example 2 (see Appendix). Figure 3 The sample exhibited excellent dispersibility. This indicates that the process utilizes the osmotic pressure difference formed by the polymer outside the dialysis membrane to drive water expulsion through a gentle concentration mechanism of reverse dialysis, avoiding vesicle aggregation caused by mechanical shear forces or chemical precipitants. Meanwhile, Zeta potential data (-1.13 mV to -3.79 mV) and biomarker detection results (see appendix) also showed excellent dispersibility. Figure 2 This further confirmed that the obtained product possesses good colloidal stability and biological activity.

[0120] Test Example 2: Comparative Evaluation of the Comprehensive Performance of Different Preparation Processes

[0121] Test method:

[0122] To verify the advantages of the technical solution of this invention in terms of yield, purity, and process efficiency, the products prepared in Example 2 (representing the best implementation of this invention) were compared with those prepared in Comparative Examples 1 to 5. The methods for measuring and calculating each indicator are as follows:

[0123] Total protein recovery and specific purity were calculated using the BCA method to determine the total protein concentration (mg / mL) of the final product for each group, combined with the particle concentration measured by NanoFCM. Specific purity is defined as the ratio of particle number to protein content. The higher the ratio, the more exosome particles per unit protein content and the less residual contaminating proteins.

[0124] The single-batch preparation cycle statistics record the cumulative operation time required from the initial treatment of cell supernatant to the final product, including centrifugation, settling, dialysis or incubation time.

[0125] The overall process performance evaluation comprehensively considers equipment requirements, consumable costs, and final product quality. The total exosome recovery is calculated based on the total number of particles after normalizing the volume of the starting material.

[0126] Test results:

[0127] The comprehensive performance test data for each group of samples are detailed in the table below.

[0128] Table 2. Performance Comparison Data of Exosome Preparation by the Invention Process and Existing Technologies

[0129] Group Final product volume (mL) Particle concentration (particles / mL) Total Particles Total protein concentration (mg / mL) <![CDATA[Specific purity (×10 8 part. / µg)]]> PdI value Preparation cycle (h) Example 2 32.5 <![CDATA[2.59×10 11 ]]> <![CDATA[8.42×10 12 ]]> 0.84 3.08 0.284 ～9.5 Comparative Example 1 (Ultracentrifugation) 2.0 <![CDATA[1.85×10 12 ]]> <![CDATA[3.70×10 12 ]]> 1.15 1.61 0.315 ～14.0 Comparative Example 2 (Ultrafiltration Tube) 30.0 <![CDATA[1.42×10 11 ]]> <![CDATA[4.26×10 12 ]]> 2.68 0.53 0.462 ～6.5 Comparative Example 3 (PEG Precipitation) 4.5 <![CDATA[3.12×10 12 ]]> <![CDATA[1.40×10 13 ]]> 14.21 0.22 0.687 ～16.0 Comparative Example 4 (without concentration) 495.0 <![CDATA[1.35×10 9 ]]> <![CDATA[6.68×10 11 ]]> 0.09 0.15 0.358 ～13.0 Comparative Example 5 (Excessive Centrifugation) 33.0 <![CDATA[4.21×10 10 ]]> <![CDATA[1.39×10 12 ]]> 0.62 0.68 0.301 ～10.5

[0130] Conclusions and Analysis

[0131] Table 2 shows the impact of different process routes on the quality of the final product. The analysis, combined with the mechanism of this invention and the accompanying figures, is as follows:

[0132] Regarding purity and impurity removal: The specific purity of Example 2 reached 3.08 × 10⁻⁶. 8 The particle / µg recovery rate is significantly better than all comparative examples. Compared with Comparative Example 3, which uses the PEG precipitation method, although its total particle recovery appears to be the highest, its protein concentration is abnormally high (14.21 mg / mL), resulting in extremely low specific purity (0.22) and a PdI value as high as 0.687, indicating severe non-specific co-precipitation. This invention utilizes reverse dialysis technology. The molecular weight cutoff (30 kD) of the dialysis membrane allows impurities smaller than this pore size to diffuse to the outside of the membrane. Simultaneously, the polymers outside the membrane only generate osmotic pressure without directly contacting the sample, thus achieving the dual effect of impurity precipitation and vesicle retention during concentration, effectively solving the problems of chemical reagent residues and impurity protein contamination.

[0133] Regarding recovery rate and structural integrity: Total particle recovery in Example 2 (8.42 × 10⁻⁶) 12 The result was significantly higher than that of Comparative Example 1 (3.70 × 10⁻⁶) obtained by ultracentrifugation. 12 NanoFCM detection data showed that the concentration in Example 2 reached 2.59 × 10⁻⁶. 11 particles / mL (as attached) Figure 1 This demonstrates the high efficiency of recovery. While ultracentrifugation is a classic method, the high centrifugal force of 100,000g can easily cause exosome structure rupture or irreversible precipitation compaction, resulting in sample loss. This invention abandons the ultracentrifugation step, controlling the centrifugal force at 10,000g to remove large particulate impurities, followed by physical concentration through a gentle osmotic pressure difference, preserving the integrity of the vesicle structure to the greatest extent.

[0134] Regarding process efficiency and dispersibility: In terms of dispersibility, the PdI value of Example 2 was only 0.284, significantly lower than that of Comparative Example 2 (0.462) and Comparative Example 3 (0.687). This low PdI value is consistent with the attached... Figure 3 The single main peak distribution observed in the samples confirms that the process of this invention avoids particle aggregation caused by mechanical shearing or chemical precipitation. Furthermore, the reverse dialysis process of this invention, while ensuring high concentration and high purity, eliminates the risk of ultrafiltration membrane clogging, and the dialysis bag throughput can be linearly scaled up, demonstrating significant advantages for industrial-scale production.

Claims

1. A method for preparing stem cell exosomes suitable for industrial-scale production, characterized in that, Includes the following steps: The stem cell culture supernatant was taken and centrifuged three times in sequence at an ambient temperature of 2℃-8℃. The supernatant was collected after each centrifugation to obtain crude exosome samples. The centrifugal force of the first stage of centrifugation is 300g-400g, the centrifugal force of the second stage of centrifugation is 1000g-3000g, and the centrifugal force of the third stage of centrifugation is 8000g-12000g. The crude exosome sample is placed into a dialysis bag, and the dialysis bag is immersed in reverse dialysis concentrate for dialysis treatment until the sample volume in the dialysis bag is concentrated to a predetermined ratio. The retentate in the bag is collected. The reverse dialysis concentrate is a buffer solution containing polyethylene glycol, and the mass-volume concentration of polyethylene glycol in the solution is 30%-50% (w / v). The retained solution was filtered through a filter membrane with a pore size of 0.1µm-0.22µm, and the filtrate was collected to obtain stem cell exosomes.

2. The method for preparing stem cell exosomes suitable for industrial-scale production according to claim 1, characterized in that, The centrifugation times for the three-stage centrifugation process are as follows: The centrifugation time for the first stage is 10-20 minutes, the centrifugation time for the second stage is 10-30 minutes, and the centrifugation time for the third stage is 20-40 minutes.

3. The method for preparing stem cell exosomes suitable for industrial-scale production according to claim 1, characterized in that, The dialysis bags are made of regenerated cellulose, with a retention capacity of 3kD-100kD.

4. The method for preparing stem cell exosomes suitable for industrial-scale production according to claim 1, characterized in that, The solvent for the reverse dialysis concentrate is a phosphate buffer solution with a molar concentration of 0.01 mol / L to 0.05 mol / L and a pH of 7.2 to 7.

6.

5. The method for preparing stem cell exosomes suitable for industrial-scale production according to claim 1, characterized in that, During dialysis, the volume ratio of the reverse dialysis concentrate to the crude exosome sample is 10:1-15:

1.

6. The method for preparing stem cell exosomes suitable for industrial-scale production according to claim 1, characterized in that, The dialysis treatment was carried out at an ambient temperature of 2℃-8℃ and a stirring speed of 100rpm-300rpm.

7. The method for preparing stem cell exosomes suitable for industrial-scale production according to claim 1, characterized in that, The dialysis treatment continues until the sample volume in the dialysis bag is concentrated to 10%-50% of its original volume, at which point it is terminated.

8. The method for preparing stem cell exosomes suitable for industrial-scale production according to claim 1, characterized in that, Before the dialysis process, the reverse dialysis concentrate is pre-filtered using a filter membrane with a pore size of 0.22µm-0.45µm.

9. The method for preparing stem cell exosomes suitable for industrial-scale production according to claim 1, characterized in that, The filter membrane is made of polyethersulfone.

10. The method for preparing stem cell exosomes suitable for industrial-scale production according to claim 1, characterized in that, The steps for obtaining the stem cell culture supernatant include: seeding human adipose-derived mesenchymal stem cells in a serum-free culture medium for culture, and collecting the stem cell culture supernatant when the cells grow to a confluence of 80%-90%.

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