Preserving fluid for stably preserving umbilical cord mesenchymal stem cell exosome for long time

Abstract

The invention provides a preserving fluid for stably preserving umbilical cord mesenchymal stem cell exosomes for a long time, which belongs to the technical field of biotechnology and comprises the following components: a mannitol solution, a dimethyl sulfoxide solution, a human serum albumin solution and beta-nicotinamide mononucleotide. The invention provides a preserving fluid for stably preserving umbilical cord mesenchymal stem cell exosomes for a long time. The number and physicochemical properties of the umbilical cord mesenchymal stem cell exosomes are protected from being affected by freezing.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology, and in particular to a preservation solution for stably and long-term preservation of umbilical cord mesenchymal stem cell exosomes. Background Art

[0002] Exosomes are a type of extracellular vesicle secreted by cells. They range in diameter from 30 nm to 150 nm and are enclosed by a phospholipid bilayer membrane. They contain a rich array of bioactive substances, including DNA, RNA, and proteins. Almost all cells, including mesenchymal stem cells (MSCs), can secrete exosomes and release them into the extracellular environment, such as cell culture supernatants and various body fluids, including blood, lymph, cerebrospinal fluid, saliva, urine, and amniotic fluid. Exosomes can deliver their contents to recipient cells, mediating intercellular communication and signal transduction, influencing the cellular microenvironment, and regulating the expression of related proteins. Studies have shown that exosomes derived from umbilical cord mesenchymal stem cells (UCMSC-Exo) exhibit similar functions to umbilical cord mesenchymal stem cells (UCMSCs), promoting the migration and proliferation of endothelial cells and fibroblasts, promoting angiogenesis and wound healing, and preventing scarring. Compared with UCMSC, UCMSC-Exo is stable in nature, easy to extract, store and transport, has low in vivo rejection, and has broad application prospects in fields such as tissue repair and regeneration.

[0003] Currently, UCMSC-Exo isolation technologies include ultracentrifugation, ultrafiltration, and immunoaffinity techniques, but suitable methods and strategies for exosome preservation remain lacking. The International Society for Extracellular Vesicles (ISEV) recommended cryopreservation of exosomes in PBS (Phosphate Buffered Saline) in its 2013 research guidelines, but removed this recommendation in its 2018 research guidelines. Because exosome concentration, physical characteristics, and biological functions can change during storage due to media, time, and temperature, a preservation medium that can stably and long-term preserve UCMSC-Exo is urgently needed.

[0004] Publication No. CN116897922B, titled "A Method for Improving Exosome Stability and Exosome Preservation Solution," discloses a method for improving exosome stability and an exosome preservation solution. The exosome preservation solution is prepared by mixing pectin and / or sodium alginate with a phosphate buffer. During use, the exosome preservation solution is first heated to form a colloidal solution, and then fresh exosomes are added. After sealing, the exosomes can be preserved for a long time at room temperature. The preservation solution can increase the charge of the exosome system, prevent changes in the particle size, concentration, and activity of the exosomes, and protect the integrity and stability of the exosomes, allowing the exosomes to maintain their biological activity for a long time and maintain the stability of their contents. However, the above technical solution only preserves exosomes at room temperature. Summary of the Invention

[0005] In view of this, the present invention provides a preservation solution for stably and long-term preservation of umbilical cord mesenchymal stem cell exosomes, thereby protecting the quantity and physicochemical properties of umbilical cord mesenchymal stem cell exosomes from the effects of freezing.

[0006] To achieve the above objectives, the present invention provides a preservation solution for stably and long-term preservation of umbilical cord mesenchymal stem cell exosomes, comprising the following components: mannitol solution, dimethyl sulfoxide solution, human serum albumin solution and β-nicotinamide mononucleotide.

[0007] Optionally, the umbilical cord mesenchymal stem cell exosome preservation solution is prepared in the following proportions: 5-7 mL of mannitol solution, 1-3 mL of dimethyl sulfoxide solution, 1-3 mL of human serum albumin solution, and 30-70 mg of β-nicotinamide mononucleotide per 10 mL.

[0008] Optionally, the umbilical cord mesenchymal stem cell exosome preservation solution is prepared in the following proportions: 6 mL of mannitol solution, 2 mL of dimethyl sulfoxide solution, 2 mL of human serum albumin solution, and 50 mg of β-nicotinamide mononucleotide per 10 mL.

[0009] Optionally, the concentration of the mannitol solution is 15%-25% (w / v).

[0010] Optionally, the concentration of the dimethyl sulfoxide solution is 90-100% (v / v).

[0011] Optionally, the concentration of the human albumin solution is 15%-25% (w / v).

[0012] Optionally, the umbilical cord mesenchymal stem cell exosome preservation solution is prepared in the following proportions: 6 mL of 20% mannitol solution, 2 mL of 99% dimethyl sulfoxide solution, 2 mL of 20% human serum albumin solution and 50 mg of β-nicotinamide mononucleotide per 10 mL.

[0013] In order to achieve the above objectives, the present invention also provides the use of the above-mentioned preservation solution for stably and long-term preservation of umbilical cord mesenchymal stem cell exosomes, wherein the preservation solution for umbilical cord mesenchymal stem cell exosomes is mixed with umbilical cord mesenchymal stem cell exosomes and stored at -70°C to -90°C.

[0014] The above technical solution of the present invention includes at least the following beneficial effects: The present invention provides a stable and long-term preservation solution for umbilical cord mesenchymal stem cell exosomes. The solution uses dimethyl sulfoxide (DMSO) to combine with water molecules to lower the freezing point, reduce mechanical damage to the exosome membrane structure caused by ice crystal formation, and inhibit water extravasation, thereby avoiding shrinkage and membrane rupture caused by exosome dehydration. Human serum albumin regulates water dynamic balance, reduces fluid exudation from the extracellular space, and reduces the risk of exosome aggregation. At the same time, it forms a protective layer through surface adsorption, reducing the physical and chemical damage to the exosomes by the external environment. Mannitol stabilizes the phospholipid structure of the exosome membrane through regulating osmotic pressure and a unique protective mechanism, preventing membrane lipid oxidation and phase transition, and maintaining the integrity of the exosome morphology and contents. β-Nicotinamide mononucleotide (NMN) enhances the metabolic activity of exosomes by activating cellular energy metabolism pathways (such as NAD+ synthesis), while scavenging free radicals, inhibiting oxidative stress reactions, and effectively delaying the loss of activity of exosomes due to oxidation during the preservation process.

[0015] The preservation solution provided by the present invention forms multi-dimensional synergistic protection through different components. The combined action and optimized ratio of DMSO, human serum albumin, mannitol and NMN can achieve long-term maintenance of the number and physicochemical properties of exosomes under ultra-low temperature freezing (-70℃~-90℃). DETAILED DESCRIPTION

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of the present invention.

[0017] Example 1 A preservation solution for stable and long-term preservation of umbilical cord mesenchymal stem cell exosomes is prepared as follows: each 10 mL contains 6 mL of 20% mannitol solution, 2 mL of 99% dimethyl sulfoxide solution, 2 mL of 20% human serum albumin solution and 50 mg of β-nicotinamide mononucleotide.

[0018] The mannitol solution was premixed with the dimethyl sulfoxide solution, and human serum albumin solution and β-nicotinamide mononucleotide were added and mixed to obtain a preservation solution, which was stored at 4°C.

[0019] Example 2 A preservation solution for stable and long-term preservation of umbilical cord mesenchymal stem cell exosomes has the following preparation ratio: each 10 mL contains 5 mL of 20% mannitol solution, 2 mL of 99% dimethyl sulfoxide solution, 3 mL of 20% human serum albumin solution and 70 mg of β-nicotinamide mononucleotide.

[0020] The mannitol solution was premixed with the dimethyl sulfoxide solution, and human serum albumin solution and β-nicotinamide mononucleotide were added and mixed to obtain a preservation solution, which was stored at 4°C.

[0021] Example 3 A preservation solution for stable and long-term preservation of umbilical cord mesenchymal stem cell exosomes has the following preparation ratio: each 10 mL contains 7 mL of 20% mannitol solution, 1 mL of 99% dimethyl sulfoxide solution, 2 mL of 20% human serum albumin solution and 30 mg of β-nicotinamide mononucleotide.

[0022] The mannitol solution was premixed with the dimethyl sulfoxide solution, and human serum albumin solution and β-nicotinamide mononucleotide were added and mixed to obtain a preservation solution, which was stored at 4°C.

[0023] Example 4 A preservation solution for stable and long-term preservation of umbilical cord mesenchymal stem cell exosomes has the following preparation ratio: 6 mL of 25% mannitol solution, 2 mL of 100% dimethyl sulfoxide solution, 2 mL of 15% human serum albumin solution and 40 mg of β-nicotinamide mononucleotide per 10 mL.

[0024] The mannitol solution was premixed with the dimethyl sulfoxide solution, and human serum albumin solution and β-nicotinamide mononucleotide were added and mixed to obtain a preservation solution, which was stored at 4°C.

[0025] Example 5 A preservation solution for stable and long-term preservation of umbilical cord mesenchymal stem cell exosomes has the following preparation ratio: each 10 mL contains 6 mL of 15% mannitol solution, 2 mL of 90% dimethyl sulfoxide solution, 2 mL of 25% human serum albumin solution and 60 mg of β-nicotinamide mononucleotide.

[0026] The mannitol solution was premixed with the dimethyl sulfoxide solution, and human serum albumin solution and β-nicotinamide mononucleotide were added and mixed to obtain a preservation solution, which was stored at 4°C.

[0027] Comparative Example 1 PBS buffer, a cryoprotectant commonly used in the laboratory for freezing stem cells, was used as the preservative.

[0028] Comparative Example 2 A cryoprotectant commonly used in the laboratory for freezing stem cells, namely a trehalose solution with a molar concentration of 25 mM, was used as a preservative.

[0029] Comparative Example 3 A cryoprotectant commonly used in the laboratory for freezing stem cells, namely a 30% volume fraction glycerol solution, was used as the preservative.

[0030] Ultra-low temperature freezing test The supernatant of P3 umbilical cord mesenchymal stem cells was selected and fresh exosome samples were prepared by ultra-high-speed centrifugation. The total number of particles was greater than 8×10 10 The exosome samples were divided equally into eight portions. Two portions of the preservation solution from Example 1 and Comparative Examples 1-3 were taken, for a total of eight portions, each containing 1 mL. The divided exosome samples were resuspended in the preservation solution and stored in an ultra-low temperature freezer at -80°C for one month. After removal, the exosome particle count was determined using a nanoparticle size analyzer. The data are shown in Table 1.

[0031] Table 1 Exosome particle concentration in the preservation solution of Example 1 and Comparative Examples 1-3

[0032] As shown in Table 1, when the preservation solution provided by the present invention preserves umbilical cord mesenchymal stem cell exosomes at ultra-low temperature, the quantity and physicochemical properties of the exosomes are stably and long-term maintained.

[0033] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A storage solution for stable and long-term storage of umbilical cord mesenchymal stem cell exosomes, characterized in that: The invention comprises the following components: mannitol solution, dimethyl sulfoxide solution, human serum albumin solution and β-nicotinamide mononucleotide.

2. The storage solution for stable and long-term storage of umbilical cord mesenchymal stem cell exosomes according to claim 1, characterized in that The umbilical cord mesenchymal stem cell exosome preservation solution is prepared in the following proportions: 5-7 mL of mannitol solution, 1-3 mL of dimethyl sulfoxide solution, 1-3 mL of human serum albumin solution, and 30-70 mg of β-nicotinamide mononucleotide per 10 mL.

3. The storage solution for stable and long-term storage of umbilical cord mesenchymal stem cell exosomes according to claim 2, characterized in that: The umbilical cord mesenchymal stem cell exosome preservation solution is prepared in the following proportions: 10 mL contains 6 mL of mannitol solution, 2 mL of dimethyl sulfoxide solution, 2 mL of human serum albumin solution, and 50 mg of β-nicotinamide mononucleotide.

4. The storage solution for stable and long-term storage of umbilical cord mesenchymal stem cell exosomes according to claim 2, characterized in that The concentration of the mannitol solution is 15%-25% (w / v).

5. The storage solution for stable and long-term storage of umbilical cord mesenchymal stem cell exosomes according to claim 2, characterized in that: The concentration of the dimethyl sulfoxide solution is 90-100% (v / v).

6. The storage solution for stable and long-term storage of umbilical cord mesenchymal stem cell exosomes according to claim 2, characterized in that: The concentration of the human albumin solution is 15%-25% (w / v).

7. The storage solution for stable and long-term storage of umbilical cord mesenchymal stem cell exosomes according to claim 2, characterized in that: The preparation ratio of the umbilical cord mesenchymal stem cell exosome preservation solution is as follows: each 10 mL contains 6 mL of 20% mannitol solution, 2 mL of 99% dimethyl sulfoxide solution, 2 mL of 20% human serum albumin solution and 50 mg of β-nicotinamide mononucleotide.

8. A use of the preservation solution for stably and long-term preservation of umbilical cord mesenchymal stem cell exosomes according to any one of claims 1 to 7, characterized in that: The umbilical cord mesenchymal stem cell exosome preservation solution is mixed with the umbilical cord mesenchymal stem cell exosomes and then stored at -70°C to -90°C.

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