Preserving fluid of porcine fat-derived mesenchymal stem cell exosome as well as preparation method and application of preserving fluid

Abstract

The invention relates to the technical field of biomedical sample preservation, and discloses a preserving fluid of porcine fat-derived mesenchymal stem cell exosomes, a preparation method and application thereof. The exosome preserving fluid provided by the invention comprises the following components in percentage by weight: 5%-20% of polyvinylpyrrolidone (PVP); 2%-10% of trehalose; 0.5%-5% of glycerol; according to the cryoprotectant, a cryoprotectant is adopted, a phosphate buffer solution (PBS) is adopted as a solvent, polyvinylpyrrolidone (PVP) is adopted to replace traditional dimethyl sulfoxide (DMSO) to serve as the cryoprotectant, the problems that in the prior art, a preserving fluid is large in toxicity and poor in preserving effect are solved, and the application of the cryoprotectant in medical external application such as wound repair, inflammation treatment and skin care is expanded.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical sample preservation, and in particular to a formulation, preparation method and application of a preservation solution specifically used to improve the stability and activity of porcine fat-derived mesenchymal stem cell exosomes during storage. Background Art

[0002] Mesenchymal stem cells (MSCs) have been widely used in regenerative medicine, immunotherapy, and the clinical treatment of various diseases due to their remarkable multipotential differentiation, immunomodulatory effects, and tissue repair capabilities. Exosomes, nanoscale vesicles secreted by MSCs that contain bioactive molecules such as proteins, lipids, and RNA, have emerged as a promising therapeutic tool. In recent years, increasing research has focused on leveraging the biological properties of MSC exosomes to promote tissue repair, immunomodulation, and anti-inflammatory effects. Within this context, preservation techniques for MSC exosomes have become an important research area, particularly with regard to ensuring their bioactivity and stability.

[0003] Porcine adipose-derived mesenchymal stem cells (PAd-MSCs) have attracted considerable attention due to their ease of access and high proliferation capacity. Porcine adipose tissue has a high fatty acid content, which positively impacts MSC growth, differentiation, and the formation and function of exosomes. Compared to stem cells from other animal sources, porcine adipose-derived MSCs exhibit immunological characteristics similar to those of human cells, offering unique advantages in immune tolerance, particularly in terms of greater suitability for transplantation and clinical applications.

[0004] Exosomes derived from porcine adipose-derived mesenchymal stem cells (MSCs) not only demonstrate potential in clinical treatment but also possess strong biological activity, enabling applications in areas such as skin repair and wound treatment. Specifically, in the field of topical medical applications, exosomes, through their inherent active molecules, can promote wound healing, reduce inflammation, and accelerate tissue regeneration. Therefore, developing a DMSO-free preservation solution that maintains the bioactivity of MSCs exosomes would not only enhance the stability of the exosomes but also ensure their efficacy in topical applications.

[0005] The use of porcine adipose-derived MSC exosomes in medical topical applications allows them to be applied directly to wound sites, releasing bioactive molecules locally and promoting healing. This technology offers the advantage of being non-invasive, avoiding the potential side effects of traditional therapies, and facilitating clinical application. To achieve this goal, the development of a porcine adipose-derived MSC exosome preservation solution must protect the exosome structure while avoiding irritation or adverse reactions to the skin and other tissues caused by ingredients such as DMSO.

[0006] Therefore, using a DMSO-free preservation solution to preserve porcine adipose-derived mesenchymal stem cell exosomes not only enhances their biological activity and stability, but also ensures their efficacy in topical medical applications, providing a new therapeutic option for regenerative medicine and wound repair. The development of this preservation solution holds significant clinical promise and lays the foundation for further promoting the use of exosomes in clinical treatments. Summary of the Invention

[0007] Traditional cell preservation solutions often use dimethyl sulfoxide (DMSO) as a cryoprotectant. While DMSO can effectively prevent freeze damage, its potential toxicity and immunogenicity to cells and exosomes during cryopreservation limits its widespread application. Therefore, research is underway to develop DMSO-free cell preservation solutions to reduce toxicity, improve safety and efficacy in clinical applications, and avoid the negative impact of DMSO on the structure and function of exosomes.

[0008] The goal of the present invention is to provide a new porcine fat-derived mesenchymal stem cell exosome preservation solution, which uses polyvinylpyrrolidone (PVP) instead of traditional dimethyl sulfoxide (DMSO) as a cryoprotectant to solve the problems of high toxicity and poor preservation effect of the preservation solution in the existing technology, and expand its application in medical external application.

[0009] To achieve this goal, the present invention provides a porcine fat-derived mesenchymal stem cell exosome preservation solution, the components of which include: 5%~20% polyvinylpyrrolidone (PVP); 2%~10% trehalose; 0.5%~5% glycerol; phosphate buffered saline (PBS) were used as solvents.

[0010] PVP is a non-toxic, low-irritation water-soluble polymer compound that has been widely used in the biomedical field in products such as drug delivery and wound dressings. PVP has no obvious toxicity at low concentrations and has no adverse reactions to cells. The molecular structure of PVP is not easy to penetrate the cell membrane, and it can prevent the formation of ice crystals by forming a protective film, thereby effectively reducing cell damage during the freezing process. Compared with DMSO, PVP does not cause drastic changes in water content inside and outside the cells, avoiding the phenomenon of cell membrane rupture caused by osmotic pressure changes caused by DMSO. Importantly, PVP has good biocompatibility and can be widely used in medical devices and biological preparations. It is suitable for long-term contact with the human body, especially in medical external application without causing irritation to the skin.

[0011] Adding 2-10% trehalose to the preservation solution is a natural sugar with antioxidant, antifreeze, and anti-dehydration properties. It stabilizes the cell membrane and protein structure during freezing and thawing. By slowing the freezing rate of water, it prevents structural damage to exosomes caused by rapid freezing of water, thus reducing exosome damage. Trehalose can slow the hydration of exosome membranes and prevent damage to the membranes, thereby maintaining the function and biological activity of exosomes.

[0012] Add 0.5-5% glycerol to the preservation solution to lower the freezing point of the solution and reduce the mechanical damage caused by freezing of extracellular water.

[0013] Phosphate buffered saline (PBS) was added to adjust the pH of the preservation solution to 7.2-7.4 to ensure the stability of the pH value of the preservation solution during low-temperature storage and thawing to avoid the impact of acid-base changes on exosomes.

[0014] In some embodiments, the concentration of polyvinylpyrrolidone (PVP) is 18% to 20%; the concentration of trehalose is 6% to 8%; and the concentration of glycerol is 1% to 2%.

[0015] In some embodiments, the preservation solution comprises the following components and concentrations: Polyvinylpyrrolidone (PVP) 192.050 g / L; Trehalose 72.525 g / L; Glycerol 12.273 g / L; Phosphate buffered saline (PBS) was used as a solvent, and the pH of the preservation solution was adjusted to 7.2-7.4.

[0016] Another aspect of the present invention further provides the use of the porcine fat-derived mesenchymal stem cell exosome preservation solution, wherein the preservation solution is used for medical external application.

[0017] In some embodiments, the preservation solution is used for wound repair, skin care, or inflammation treatment.

[0018] Another aspect of the present invention also provides a method for preserving exosomes using the porcine fat-derived mesenchymal stem cell exosome preservation solution, wherein the exosomes are preserved at -80°C or lower using the preservation solution to ensure the activity and function of the exosomes.

[0019] Another aspect of the present invention further provides a method for preparing the porcine fat-derived mesenchymal stem cell exosome preservation solution, the method comprising the following steps: Step 1: Take a certain amount of phosphate buffered saline (PBS) as a solvent; add polyvinylpyrrolidone (PVP) to adjust its concentration between 5-20% and stir until completely dissolved; Step 2: Add 2-10% trehalose and 0.5-5% glycerol to the solution and continue stirring until all components are completely dissolved; Step 3: Use an ultrasonic oscillator to degas the solution to remove bubbles and prevent ice crystals from forming during freezing. Step 4: Filter the mixed exosome preservation solution through a 0.22 μm sterile filter membrane to ensure sterility and remove possible microbial contamination.

[0020] In some embodiments, the mass concentration of polyvinylpyrrolidone in step 1 is adjusted to between 18% and 20%, and 6% to 8% of trehalose and 1% to 2% of glycerol are added in step 2.

[0021] In some embodiments, the mass concentration of polyvinylpyrrolidone in step 1 is 192.050 g / L, the mass concentration of trehalose in step 2 is 72.525 g / L, and the mass concentration of glycerol is 12.273 g / L.

[0022] In some embodiments, the method further comprises step 5: packaging the filtered preservation solution into sterile cryopreservation tubes.

[0023] Compared with the prior art, the advantages and positive effects of the present invention are: Traditional exosome preservation solutions often use dimethyl sulfoxide (DMSO) as a cryopreservative. While DMSO effectively prevents ice crystal formation, it is cytotoxic and skin irritating, posing safety risks in clinical applications. By replacing DMSO with polyvinylpyrrolidone (PVP), the present invention successfully reduces the toxicity of the preservation solution, making it safer. PVP's non-toxic and low-irritation properties make it suitable not only for traditional exosome preservation but also for topical medical applications such as wound repair, inflammation treatment, and skin care. Because PVP replaces DMSO, the preservation solution's low toxicity allows it to be applied directly to wound sites or skin surfaces, avoiding the potential adverse reactions to wounds and skin caused by traditional DMSO. Exosomes, as natural bioactive carriers, play a significant role in wound healing and anti-inflammatory effects. The preservation solution of this invention ensures that exosomes can fully exert their desired biological functions after resuscitation, expanding the application of exosomes.

[0024] Traditional DMSO preservation solutions require additional processing steps during use and storage, are complex to operate, and pose potential safety risks. However, the present invention utilizes natural, non-toxic ingredients such as PVP, reducing both operational complexity and costs, and simplifying the storage and application of exosomes. This preservation solution offers improved solubility and ease of use, making the storage and clinical application of exosomes more convenient and economical. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 is the Pareto chart of standardized effects; Figure 2 The optimal level of each factor when the cell survival rate of the response amount reaches the maximum value; Figure 3 The exosome morphology (TEM) at day 0, month 3, and month 6 of cryopreservation is shown; Figure 4 The exosome particle size (NAT) at day 0, month 3, and month 6 of cryopreservation; Figure 5A This is the flow cytometry result of exosomes on day 0 of cryopreservation. Figure 5B This is the exosome flow cytometry result at month 3 (5B). Figure 5C The exosome flow cytometry results at month 6 (5C) are shown. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0028] The present invention minimizes the toxicity of the preservation solution while ensuring the activity of exosomes. The specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the present invention.

[0029] Example 1 Extraction of exosomes Porcine adipose-derived mesenchymal stem cells (MSCs) are selected as they have strong differentiation potential and are suitable for exosome extraction. MSCs are seeded in appropriate culture flasks or dishes and passaged and expanded when they reach 70-80% confluence. Exosome collection begins when cells reach the logarithmic growth phase and 80-90% confluence.

[0030] When cells reach logarithmic growth, remove the original culture medium (containing FBS), as FBS contains a large number of exosomes, which can interfere with the extraction process. Replace the original culture medium with serum-free medium and continue culturing for 24-48 hours to ensure exosome secretion. After 24-48 hours, collect the supernatant for exosome extraction. Centrifuge the cell culture medium to remove cells and cell debris, ensuring that the collected medium is free of cellular impurities.

[0031] After removing cells and debris, the supernatant is centrifuged at medium speed (1000g for 30 minutes) to remove larger extracellular vesicles and microparticles (such as small particles and dead cells). The supernatant is then transferred to an ultracentrifuge tube and separated by ultracentrifugation at 100,000g for 1 hour. The pellet is removed and washed with PBS to remove excess centrifugation medium and low-molecular-weight substances, thereby obtaining purified exosomes.

[0032] Example 2 Determination of the exosome preservation solution formula based on the central composite design method In order to evaluate the toxicity of the preservation solution, human keratinocytes (HaCaT cells) were selected as the experimental model. HaCaT cells are widely used in skin toxicity and repair research, and can reflect the potential effects of exosome preservation solution on the skin. The optimization goal is to minimize the toxicity of the exosome preservation solution, and the toxicity of the preservation solution is measured by cell survival rate as the response variable. The present invention adopts the central composite design (CCD) in the DoE design method, which can effectively analyze the interaction and quadratic effects between the three factors (PVP concentration, trehalose concentration, and glycerol concentration). Minitab18 is used to generate a plan based on the central composite design. This plan will include central point experiments, star point experiments, and corner point experiments. The generated experimental table will list the factor combination of each experimental point. Cell culture experiments were performed according to the factor combination of each experimental point in the design table, and the cytotoxicity data of each experimental point were recorded. The response variable data of each experiment were entered into Minitab18 (Table 1), a response surface design was created, and a Pareto chart of the standardized effect was obtained ( Figure 1) and a method analysis table (Table 2) were used to assess the impact of each factor on toxicity. The standardized effect plots indicate that the linear terms of PVP concentration and glycerol concentration, as well as the squared terms of PVP concentration, trehalose concentration, and glycerol concentration, significantly affect cell viability. The analysis of variance results showed that the model showed no significant lack of fit, but a significant regression. Furthermore, the adjusted coefficient of determination (R) of the model was 0.926, with a standard deviation of regression (S) of 2.471, indicating a good fit of the regression equation and the model can be used for theoretical prediction of exosome preservation solution formulations. The experimental results were subjected to multiple regression analysis. The quadratic polynomial regression model equation was: cell viability = 28.45 + 4.321 PVP concentration + 6.75 trehalose concentration + 10.69 glycerol concentration - 0.1098 PVP concentration * PVP concentration - 0.4135 trehalose concentration * trehalose concentration - 1.350 glycerol concentration * glycerol concentration - 0.0216 PVP concentration * trehalose concentration - 0.1570 PVP concentration * glycerol concentration - 0.286 trehalose concentration * glycerol concentration. Minitab was used to optimize and solve the regression equation. When the response quantity cell viability reached the maximum value, the optimal level of each factor ( Figure 2 ): When the PVP concentration is 192.050 g / L, the trehalose concentration is 72.525 g / L, and the glycerol concentration is 1.2273 g / L, the preservation solution has the least toxicity to the cells, that is, the cell survival rate is the highest, which is 99.9829%.

[0033] Table 1 Experimental groups and response results of the central composite design method for exosome preservation solution

[0034] Table 2 Central composite design results ANOVA results

[0035] Example 3 Preparation and toxicity verification of exosome preservation solution According to the formula obtained in Example 2, 19.2050g PVP and 7.2525g trehalose were weighed respectively, dissolved in PBS, and a magnetic stirrer was used to accelerate dissolution to ensure complete dissolution. Glycerol is a liquid, so 1.2273g glycerol is converted to 0.973ml of liquid. 0.973ml of glycerol was measured and mixed with the above solution, and PBS was added to a total volume of 100ml. The solution was thoroughly mixed using a magnetic stirrer to ensure that each component was evenly distributed. Finally, the preservation solution was filtered using a 0.22um sterile filter membrane.

[0036] After the preparation of the preservation solution was completed, the CCK8 method was used to detect the toxicity of the preservation solution on HaCaT cells, and the cell survival rate was 99.53%, which was basically consistent with the predicted value, proving that the regression model is applicable.

[0037] Example 4 Exosome morphology examination The exosomes extracted in Example 1 were preserved using the exosome preservation solution prepared in Example 3, and the exosome morphology was examined at day 0, month 3, and month 6 of cryopreservation. The exosome morphology at the three time periods was observed using a transmission electron microscope (TEM). Figure 3 ), no obvious difference in morphology was found, and there was no excessive fragmentation or aggregates.

[0038] Nanoparticle tracking analysis (NTA) was used to measure the size distribution and concentration of exosomes. Figure 4 ), the results showed that there was no significant decrease in particle size distribution and concentration at day 0, month 3, and month 6.

[0039] Example 5 Exosome activity detection The stored exosomes were taken out on day 0, month 3, and month 6, and the activity of exosome CD105, CD73, and CD90 proteins was detected by flow cytometry. The results showed that there was no significant decrease in the activity of exosomes in the storage solution (Figure 5).

[0040] In summary, the preservation solution of the present invention significantly reduces toxicity during cryopreservation by using polyvinylpyrrolidone (PVP) instead of traditional dimethyl sulfoxide (DMSO) as a cryoprotectant, while effectively maintaining the biological activity and stability of exosomes. This alternative approach improves the safety of exosome preservation and expands its clinical application, particularly for various clinical scenarios such as topical medical applications, wound repair, inflammation treatment, and skin care. While traditional DMSO effectively preserves the structure and function of exosomes during cryopreservation, its cytotoxicity and skin irritation pose certain risks in clinical applications, especially when used directly in topical applications such as wound repair and skin care, potentially causing adverse reactions. By using PVP as a cryoprotectant, the present invention successfully reduces the toxicity of the preservation solution and avoids the potential cell damage and skin discomfort caused by DMSO. Furthermore, the structure and function of exosomes can be easily damaged during cryopreservation, affecting their efficacy in clinical treatment. By optimizing the preservation solution formulation, particularly by using PVP as an alternative cryoprotectant, the stability of exosomes can be significantly improved, ensuring the persistence of their biological activity during preservation and recovery. Therefore, the preservation solution of the present invention not only ensures the activity of exosomes, but also enhances their stability during long-term storage and reduces the loss of biological activity due to the freezing process. In summary, unlike traditional exosome preservation methods, the preservation solution of the present invention does not contain toxic DMSO components, making the preservation solution safer when directly applied to medical external application scenarios such as wound repair, inflammation treatment and skin care. As a natural bioactive carrier, exosomes have significant therapeutic effects in wound healing, skin repair and anti-inflammatory effects, especially in the treatment of chronic inflammation, promotion of tissue repair and anti-aging. Through the preservation solution of the present invention, exosomes can be applied to the wound site in a low-toxic and gentle manner, helping patients heal faster and effectively reducing inflammatory responses.

[0041] Although some specific embodiments of the present invention have been described in detail by way of example, it should be understood by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It should also be understood by those skilled in the art that various modifications may be made to the embodiments without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A porcine fat-derived mesenchymal stem cell exosome storage solution, characterized in that: The components of the preservation solution include: 18%~20% polyvinylpyrrolidone (PVP); 6%~8% trehalose; 1%~2% glycerol; phosphate buffered saline (PBS) were used as solvents.

2. The porcine adipose-derived mesenchymal stem cell exosome storage solution according to claim 1, wherein The preservation solution contains the following components and concentrations: Polyvinylpyrrolidone (PVP) 192.050 g / L; Trehalose 72.525 g / L; Glycerol 12.273 g / L; Phosphate buffered saline (PBS) was used as a solvent, and the pH of the preservation solution was adjusted to 7.2-7.

4.

3. Use of the porcine fat-derived mesenchymal stem cell exosome preservation solution according to any one of claims 1-2, characterized in that: The preservation solution is used for external medical application.

4. The use according to claim 3, characterized in that The preservation solution is used for wound repair or skin care.

5. A method for preserving exosomes using the porcine adipose-derived mesenchymal stem cell exosome preservation solution according to any one of claims 1 to 2, characterized in that: The exosomes are stored at -80°C or lower using the preservation solution to ensure the activity and function of the exosomes.

6. A method for preparing the porcine adipose-derived mesenchymal stem cell exosome preservation solution according to claim 1, characterized in that: The method comprises the following steps: Step 1: Take a certain amount of phosphate buffered saline (PBS) as a solvent; add polyvinylpyrrolidone (PVP) to adjust its concentration between 5-20% and stir until completely dissolved; Step 2: Add 2-10% trehalose and 0.5-5% glycerol to the solution and continue stirring until all components are completely dissolved; Step 3: Use an ultrasonic oscillator to degas the solution to remove bubbles and prevent ice crystals from forming during freezing. Step 4: Filter the mixed exosome preservation solution through a 0.22 μm sterile filter membrane to ensure sterility and remove possible microbial contamination.

7. The method according to claim 6, wherein The mass concentration of polyvinylpyrrolidone in step 1 is adjusted to between 18% and 20%, and 6% to 8% trehalose and 1% to 2% glycerol are added in step 2.

8. The method according to any one of claims 6 to 7, characterized in that The mass concentration of polyvinyl pyrrolidone in step 1 is 192.050 g / L, the mass concentration of trehalose in step 2 is 72.525 g / L, and the mass concentration of glycerol is 12.273 g / L.

9. The method according to claim 8, wherein The method further comprises step 5: packaging the filtered preservation solution into sterile cryopreservation tubes.

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