TWI896262B - Composition comprising platelet-derived exosomes and mesenchymal stem cell-derived exosomes and producing method and use thereof - Google Patents

Abstract

Provided is a composition and producing method and use thereof. The composition comprises platelet-derived exosomes and mesenchymal stem cell-derived exosomes. The composition of the present invention can reduce inflammation, promote collagen production and increase dermal fibroblast proliferation, which result in improved wound healing effect. Therefore, the composition of the present invention can be used for skin repairing.

Description

Composition containing mesenchymal stem cell exosomes and platelet exosomes, and preparation method and use thereof

The present invention relates to a composition containing multiple exosomes, a method for preparing the composition, and its use in anti-inflammation, skin repair, and hair growth.

Inflammation refers to a series of physiological reactions that occur in biological tissues after being stimulated by trauma or pathogenic infection, including redness, swelling, fever, and pain. Since inflammation is one of the body's responses to external pathogens and is a type of immune response, it is beneficial to the organism. However, some diseases can cause the body to be in a state of inflammation for a long time, also known as "chronic inflammation", such as hay fever, periodontitis, and rheumatoid arthritis. Generally speaking, chronic inflammation may last for several months to several years; in contrast, acute inflammation is an immediate reaction of tissues or organs to external stimuli, and occurs within a shorter period of time, such as minutes to days.

Inflammation also plays a crucial role in wound healing. Prolonged inflammation can make wound healing more difficult and lead to scarring. Therefore, controlling inflammation can promote local wound healing.

Sensitive skin is characterized by a thin skin barrier and low defenses, making it prone to overreaction. This type of skin can trigger inflammation due to seasonal changes, sun exposure, skincare products, or cosmetics. Due to the skin's inherent sensitivity, medications are difficult to treat. Therefore, there is an urgent need for the development of effective anti-inflammatory drugs.

In view of the needs of the existing technology, the present invention provides a composition that can reduce inflammatory reactions, promote collagen secretion, promote fibroblast proliferation, and promote wound healing. Therefore, it can be used for skin repair, especially for sensitive skin.

To achieve the aforementioned objectives, the present invention provides a composition comprising: mesenchymal stem cell exosomes and platelet exosomes.

Preferably, the mesenchymal stem cell exosomes are secreted from mesenchymal stem cells, and the mesenchymal stem cells are mesenchymal stem cells isolated from the umbilical cord, mesenchymal stem cells from the placenta, mesenchymal stem cells from the dental pulp, mesenchymal stem cells from the bone marrow, adipose stem cells from fat, hair follicle stem cells from hair follicles, or amniotic fluid stem cells from amniotic fluid.

Preferably, the platelet exosomes are secreted from platelets, and the platelets are isolated from adult peripheral blood or umbilical cord blood.

Preferably, the mesenchymal stem cell exosomes or platelet exosomes contain microRNAs (miRNAs). In one embodiment, the miRNAs are selected from hsa-let-7c-5p, hsa-miR-100-5p, hsa-miR-1246, hsa-miR-125b-5p, hsa-miR-126-3p, hsa-miR-145-5p, hsa-miR-15a-5p, hsa-miR-192-5p, hsa-miR-19b -3p, hsa-miR-21-5p, hsa-miR-221-3p, hsa-miR-23a-5p, hsa-miR-24-3p, hsa-mir-29a, hsa-miR-31-5p, hsa-miR-3665, hsa-miR-663a and hsa-miR-762, which can further promote the effect of skin repair. In another embodiment, the miRNA is selected from the group consisting of hsa-miR-103a-3p, hsa-miR-107, hsa-miR-125b-5p, hsa-miR-130b-3p, hsa-miR-140-5p, hsa-miR-146a-5p, hsa-miR-181a-5p, hsa-miR-195-5p, hsa-miR -19a-3p, hsa-miR-20a-5p, hsa-miR-22-3p, hsa-miR-22-5p, hsa-miR-335-5p, hsa-miR-34a-5p, hsa-miR-371b-5p, hsa-miR-433-3p and hsa-miR-493-3p, which can further promote hair growth. In another embodiment, the miRNA is selected from the group consisting of hsa-let-7b, hsa-let-7i-5p, hsa-miR-125b-5p, hsa-miR-142-5p, hsa-miR-145, hsa-miR-146a-5p, hsa-miR-150-3p, hsa-miR-155, hsa-miR-155-5p, hsa-miR-15a, hsa-miR-17-5p, hsa-miR-181b-5p, hsa-miR-182, hsa-miR-183-5p, hsa-miR-186, hsa-miR-18a-3p, hsa-miR-19a-3p, hsa-mi hsa-miR-19b-3p, hsa-miR-20a-5p, hsa-miR-214-3p, hsa-miR-21-5p, hsa-miR-222-3p, hsa-mir-223, hsa-miR-23a, hsa-miR-23a-3p, hsa-miR-24-2, hsa-miR-26b-5p, hsa-mir-29a, hsa-miR-29a-3p, hsa-miR-30b, hsa-miR-31-5p, hsa-miR-326, hsa-miR-330-3p, hsa-miR-424 and hsa-miR-744-5p, which can further promote the anti-inflammatory effect.

To achieve the aforementioned objectives, the present invention further provides a method for preparing the composition, comprising: Step (A): culturing mesenchymal stem cells in minimal essential medium α for 30 to 60 hours, centrifuging the resulting culture medium to remove cells and cell debris, and then concentrating and filtering the culture medium to obtain the mesenchymal stem cell exosomes; Step (B): centrifuging blood to remove the red blood cell layer to obtain a plasma layer, centrifuging the plasma layer to remove the red blood cells to obtain a platelet concentrate, subjecting the platelet concentrate to a freeze-thaw cycle to remove cell debris and obtain the platelet exosomes; and Step (C): Mixing the mesenchymal stem cell exosomes with the platelet exosomes to obtain the composition.

In one embodiment, step (A) is to obtain a solution containing mesenchymal stem cell exosomes; step (B) is to obtain a solution containing platelet exosomes; and in step (C), the mixing of the mesenchymal stem cell exosomes and the platelet exosomes is performed by mixing the solution containing mesenchymal stem cell exosomes with the solution containing platelet exosomes at a volume ratio of 1:0.1 to 1:10. Preferably, the mixing of the mesenchymal stem cell exosomes and the platelet exosomes in step (C) is performed by mixing the solution containing mesenchymal stem cell exosomes with the solution containing platelet exosomes at a volume ratio of any ratio between 1:0.2 and 1:7. For example, the aforementioned volume ratio may be 1:0.5, 1:1, 1:3, 1:5, or 1:7. In one embodiment, the solution containing mesenchymal stem cell exosomes contains 1×10 ¹⁰ exosomes per milliliter. In one embodiment, the solution containing platelet exosomes contains 5.6×10 ¹¹ to 5.8×10 ¹² exosomes per milliliter. Preferably, the particle number ratio of the mesenchymal stem cell exosomes to the platelet exosomes is 1:10 to 1:10,000. For example, the ratio may be 1:20, 1:150, 1:300, 1:500, 1:1000, 1:3000, or any ratio between 1:50 and 1:5000. In one embodiment, the particle number ratio of the mesenchymal stem cell exosomes to the platelet exosomes is 1:100 to 1:2000.

Preferably, the incubation time in step (A) is 40 to 50 hours, for example 42, 44, 46 or 48 hours.

Preferably, the culture medium obtained by culturing in step (A) is centrifuged at a relative centrifugal force of 2000×g to 3000×g for 5 to 20 minutes. For example, the centrifugation speed may be 2200×g, 2400×g, 2600×g, or 2800×g. The centrifugation time may be 7, 10, 15, or 18 minutes.

Preferably, the concentration and filtering in step (A) are performed using a concentration tube.

Preferably, the centrifugation of the blood in step (B) is performed by treating the blood with an anticoagulant. More preferably, the anticoagulant is a citrate phosphate dextrose adenine (CPDA-1) solution.

Preferably, in step (B), the blood is centrifuged to remove the red blood cell layer to obtain the plasma layer by centrifuging the blood at a relative centrifugal force of 100×g to 800×g for 5 to 40 minutes. The centrifugation time can be 7, 10, 15, 20, 25, 30, or 35 minutes.

Preferably, in step (B), the plasma layer is centrifuged to obtain a platelet concentrate. Specifically, the plasma layer is centrifuged at a relative centrifugal force of 800×g to 5000×g for 5 to 40 minutes. The centrifugation time can be 7, 10, 15, 20, 25, 30, or 35 minutes.

Preferably, the freeze-thaw cycle in step (B) is performed by freezing the platelet concentrate at a temperature of -200°C to -80°C and then rewarming it at 25°C to 40°C, and repeating this cycle two or more times, for example, three, four, five, or six times.

Preferably, in step (B), "subjecting the platelet concentrate to a freeze-thaw cycle to remove cell debris" involves removing cell debris by centrifugation and filtration. More preferably, the filtration is performed using a filter with a pore size of 0.22 μm to remove cell debris and microorganisms, thereby achieving a higher purity of the platelet exosomes. The centrifugation is performed at a relative centrifugal force of 2000 × g to 3000 × g for 5 to 20 minutes, for example, at 2200 × g, 2400 × g, 2600 × g, or 2800 × g for 13, 15, 17, or 19 minutes, respectively.

To achieve the aforementioned objectives, the present invention further provides a use of the aforementioned composition for preparing a medicament for treating, preventing, or alleviating inflammatory diseases. Preferably, the inflammatory disease comprises rheumatoid arthritis.

The present invention demonstrates that the composition of the present invention can effectively promote collagen production. Therefore, to achieve the aforementioned objectives, the present invention further provides a use of the aforementioned composition for skin repair.

Preferably, the skin is sensitive skin.

The present invention demonstrates that the composition of the present invention can promote fibroblast proliferation and collagen production. To achieve the aforementioned objectives, the present invention further provides a use of the aforementioned composition for preparing a medicament for promoting wound healing.

To achieve the aforementioned objectives, the present invention further provides a use of the aforementioned composition for promoting hair growth. According to the present invention, the composition can promote fibroblast proliferation. Therefore, when applied to the scalp, it can promote the proliferation of fibroblasts in hair follicles, thereby promoting hair follicle regeneration, hair growth, and improving graying hair.

The composition of the present invention can reduce the expression of interferon gamma (IFN-γ) in peripheral blood mononuclear cells induced by phytohemagglutinin, thereby reducing inflammatory responses. It can also promote collagen secretion and fibroblast proliferation, thereby promoting wound healing. Therefore, it can be used for skin repair, especially for sensitive skin. It can also promote hair growth.

The following, in conjunction with the drawings, preparation examples, and experimental examples of the present invention, further illustrates the technical means adopted by the present invention to achieve the predetermined purpose of the invention.

Example 1 Preparation of Mesenchymal Stem Cell Exosomes

Mesenchymal stem cells isolated from the umbilical cord were cultured in complete culture medium containing Minimum Essential Medium α (α-MEM) (Gibco cat#41061037), 5% to 10% human platelet lysate, 1% L-glutamine, 1% non-essential amino acids (Gibco cat#11140050), 1% sodium pyruvate, and 1% penicillin-streptomycin. Mesenchymal stem cells were grown in an incubator at 37°C and 5% _CO₂ . When the cells reached 80% to 90% confluency, the culture medium was replaced with α-MEM medium without additives and cultured for an additional 48 hours to induce exosome release. The culture medium was removed and centrifuged at 2330 × g for 10 minutes to remove cellular debris. The exosomes were then concentrated using a concentrator (Macrosep UF Centrifugal Devices, MWCO 3K) to obtain a solution containing mesenchymal stem cell exosomes. Using a nanoparticle tracking analyzer, exosomes were labeled with exosome markers CD9, CD63, and CD81, and the number of exosomes in the sample was analyzed. The solution containing mesenchymal stem cell exosomes was found to contain 1×10 ¹⁰ exosomes per milliliter (mL), which was used in subsequent examples.

Example 2 Preparation of Platelet Exosomes

Adult peripheral blood stored in a CPDA-1 blood bag was centrifuged at a relative centrifugation force of 200×g to 500×g for 10 to 30 minutes to separate into a plasma layer and an erythrocyte layer. The lower erythrocyte layer was removed, and the upper plasma layer was retained. The plasma layer was further centrifuged at a relative centrifugation force of 1000×g to 4000×g for 10 to 30 minutes to obtain a platelet concentrate. The platelet concentrate was then subjected to three freeze-thaw cycles to rupture the platelets and filtered through a 0.22 μm filter to remove cellular debris and microorganisms, resulting in a solution containing platelet exosomes. Using a nanoparticle tracking analyzer, exosomes were labeled with exosome markers CD9, CD63, and CD81, and the number of exosomes in the sample was analyzed. The platelet-exosome solution contained 5.6×10 ¹¹ to 5.8×10 ¹² exosomes per milliliter. The platelet-derived growth factor (PDGF-BB) protein concentration in the platelet-exosome solution was measured using the human platelet-derived growth factor (PDGF-BB) kit (R&D systems cat#DBB00), confirming that the platelet-exosomes were rich in growth factor.

Test Example 1: Anti-inflammatory test

Peripheral blood mononuclear cells (PBMCs) isolated from adult peripheral blood were cultured at 4×10 ⁵ cells/mL in minimal essential medium α in a 24-well plate. Phytohemagglutinin (PHA) was added to the culture medium to a concentration of 10 μg/ml was used to induce inflammatory response in PBMC cells. After adding the aforementioned PHA, the PBMC cells were immediately subjected to the following treatments: the mesenchymal stem cell exosomes obtained in Example 1, the platelet exosomes obtained in Example 2, and a combined treatment group of platelet exosomes and mesenchymal stem cell exosomes (hereinafter referred to as the combined treatment group). The combined treatment was performed by taking 0.25 mL of the mesenchymal stem cell exosome solution obtained in Example 1 and 0.75 mL of the platelet exosome solution obtained in Example 2. The mesenchymal stem cell exosome solution contained 1×10 ¹⁰ mesenchymal stem cell exosomes per mL; the platelet exosome solution contained 5.6×10 ¹¹ to 5.8 × 10 ¹² exosomes. PBMCs were treated with 0.5 mL of each treatment group for 72 hours. PBMCs treated with PHA alone served as a control group.

The IFN-γ level in each group, as an inflammatory marker, was then measured in the following manner: 100 μL of the culture medium co-cultured with each treatment group and the control group, as well as 100 μL of the prepared IFN-γ standard, were added to a 96-well plate containing a Human IFN-gamma ELISA Kit (RayBiotech cat#ELH-IFNg-1) for a test reaction. The reaction was gently shaken at room temperature for 2.5 hours. The solution was removed and the plates were washed with wash buffer. The detection antibody anti-IFN-γ in the kit was then added and the reaction was gently shaken at room temperature for 1 hour. All the solution in the wells was removed and washed with wash buffer. The horseradish peroxidase marker was then added. The plate was incubated with a horseradish peroxidase-Streptavidin (HRP-Streptavidin) solution and gently shaken at room temperature for 45 minutes. The solution was removed and washed with wash buffer. 3,3',5,5'-tetramethylbenzidine (TMB) reagent (provided in the kit) was then added and gently shaken at room temperature in the dark for 30 minutes. The Stop solution (provided in the kit) was added and the absorbance at 450 nm was immediately measured using a microplate analyzer. The IFN-γ concentration-to-absorbance conversion equation was calculated based on the standard solution concentration and the average absorbance. The IFN-γ concentration in each treatment group and control group was calculated using the aforementioned IFN-γ concentration conversion equation. The concentration of the control group was set as 100%, and the percentage of IFN-γ concentration in each treatment group relative to that of the control group was calculated.

The test results, shown in Figure 1, show that treatment with MSC exosomes reduced IFN-γ secretion by peripheral blood monocytes by 40% compared to the control group. Platelet exosome treatment also demonstrated a similar effect, with a 38% reduction compared to the control group. However, when platelet exosomes and MSC exosomes were combined, the reduction in IFN-γ secretion by peripheral blood monocytes was even more significant, reaching a 71% reduction compared to the control group. The IFN-γ-reducing effect was unexpected, as the combined treatment group contained only 25% MSC exosomes and 75% PBMC exosomes. Therefore, the combined treatment group should have only reduced IFN-γ secretion by 38.5% (40% × 25% + 38% × 75%). Therefore, the combined treatment group had a multiplicative and synergistic effect in reducing IFN-γ secretion by peripheral blood mononuclear cells. Because the combined treatment group showed superior results, subsequent experiments were conducted using the combined treatment group, with a fixed volume ratio of MSC exosome solution to platelet exosome solution of 1:3.

Test Example 2: Wound Healing Simulation Test

Normal human dermal fibroblasts (NHDF) (hereinafter referred to as NHDF cells) were seeded at a cell density of 2×10 ⁵ per well in a 6-well plate in Minimum Essential Medium α (α-MEM) (Gibco cat#11140050) supplemented with 5% to 10% human platelet lysate, 1% L-glutamine, 1% non-essential amino acids, and 1% sodium pyruvate . The plates were cultured in complete medium (cat#41061037) for 48 hours to allow cells to attach and form a cell layer. A cell-free zone approximately 0.5 mm to 1 mm in width was then scraped from the culture dish. The plates were then washed with PBS to remove detached cells and debris. NHDF cells were then treated with a 2 mL volume of platelet exosomes and mesenchymal stem cell exosomes combined and co-cultured for 24 hours. For the control group, NHDF cells were cultured in minimal essential medium α for the corresponding combined treatments. Finally, the wound healing enhancement results of each group were observed under a microscope.

The results are shown in Figure 2. The center of the photo shows the area where cells were scraped. It can be seen that the combined treatment group significantly promoted wound healing. Therefore, the composition of the present invention can be used to promote wound healing.

Test Example 3 Cell proliferation test

NHDF cells were seeded at a density of 2000 cells per well in 96-well plates in Minimum Essential Medium α (α-MEM) (Gibco cat#11140050) supplemented with 5% to 10% human platelet lysate, 1% L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, and 1% penicillin-streptomycin . Cells were cultured in complete culture medium (cat#41061037) for 48 hours to allow uniform attachment to the plate bottom. After removing the cell suspension, the following treatments were performed: NHDF cells were treated with 0.1 mL of a combination of platelet exosomes and mesenchymal stem cell exosomes and co-cultured with the cells for 72 hours; in the control group, NHDF cells were cultured with minimal essential medium α for the corresponding combined treatments. The culture medium was then replaced with a medium containing MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) cell viability reagent and incubated at 37°C for 3 hours. The color reaction was detected using an enzyme immunoassay (ELISA) with an absorbance reading at 490 nanometers (nm), and the cell viability percentage was calculated. The results are shown in Figure 3.

The results in Figure 3 show that the combined treatment group, because it treated cells with both platelet exosomes and stem cell exosomes, significantly promoted NHDF cell proliferation compared to the control group over the same period of time. This indicates that combined treatment with platelet exosomes and stem cell exosomes can accelerate NHDF cell proliferation. Therefore, the composition of the present invention can be used to promote wound healing.

Test Example 4: Collagen Secretion Test

NHDF cells were seeded at a density of 2000 cells per well in a 96-well plate and cultured in Minimum Essential Medium α (α-MEM) (Gibco cat#41061037) supplemented with 5% to 10% human platelet lysate, 1% L-glutamine, 1% non-essential amino acids (Gibco cat#11140050), 1% sodium pyruvate, and 1% penicillin-streptomycin for 48 hours . The cells were uniformly attached to the plate bottom. After the cell suspension was removed, 0.1 mL of platelet exosomes and mesenchymal stem cell exosomes were co-cultured with NHDF cells for 72 hours. For the control group, NHDF cells were cultured with minimal essential medium α. The collagen content secreted by NHDF cells was then measured using the Procollagen Type I –peptide (PIP) EIA Kit (TaKaRa cat#MK101) coupled with an enzyme-linked immunosorbent assay. The results are shown in Figure 4.

The results in Figure 4 show that the combined treatment group, because the cells were treated with both platelet exosomes and stem cell exosomes, significantly increased the amount of collagen secreted by NHDF cells compared to the control group over the same period of time. Therefore, the composition of the present invention can be used to promote wound healing.

In summary, the composition of the present invention, because it contains both mesenchymal stem cell exosomes and platelet exosomes, can reduce inflammation, promote collagen secretion, and promote fibroblast proliferation, thereby promoting wound healing. Therefore, it can be used for skin repair, especially for sensitive skin.

Various modifications and variations of the present invention will be readily apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described with reference to certain preferred embodiments, it should be understood that the present invention should not be unduly limited to those specific embodiments. In fact, various modifications that would be obvious to those skilled in the art in practicing the described modes of the invention are also intended to be covered by the following patent applications.

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Figure 1 shows IFN-γ production by NHDF cells treated with induced inflammation, including mesenchymal stem cell exosomes, platelet exosomes, or a combination of platelet exosomes and mesenchymal stem cell exosomes. Figure 2 shows the wound healing promotion results of NHDF cells treated with these combinations and a control group. Figure 3 shows the cell proliferation rate of NHDF cells treated with these combinations and a control group. Figure 4 shows the collagen secretion of NHDF cells treated with these combinations and a control group.

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Claims (11)

A composition comprising: mesenchymal stem cell exosomes and platelet exosomes, wherein the particle number ratio of the mesenchymal stem cell exosomes to the platelet exosomes is 1:100 to 1:2000. The composition of claim 1, wherein the mesenchymal stem cell exosomes are secreted from mesenchymal stem cells, and the mesenchymal stem cells are isolated from umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, dental pulp mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose stem cells, hair follicle stem cells, or amniotic fluid stem cells. The composition of claim 1, wherein the platelet exosomes are secreted from platelets, and the platelets are isolated from adult peripheral blood or umbilical cord blood. The composition of claim 1, wherein the mesenchymal stem cell exosomes or platelet exosomes contain microRNA. A method for preparing the composition of any one of claims 1 to 4, comprising: step (A): culturing mesenchymal stem cells in a minimum essential medium α for 30 to 60 hours, centrifuging the culture medium obtained from the culture to remove cells and cell debris, and then concentrating and filtering to obtain the mesenchymal stem cell exosomes; step (B): centrifuging blood to remove the red blood cell layer to obtain a plasma layer, then centrifuging the plasma layer to remove the red blood cells to obtain a platelet concentrate, subjecting the platelet concentrate to a freeze-thaw cycle to remove cell debris, and obtaining the platelet exosomes; and Step (C): mixing the mesenchymal stem cell exosomes with the platelet exosomes to obtain the composition. A use of the composition according to any one of claims 1 to 4 for preparing a medicament for treating, preventing or alleviating inflammatory diseases. The use of the composition as described in claim 6, wherein the inflammatory disease comprises rheumatoid arthritis. A use of the composition according to any one of claims 1 to 4, for preparing a medicament for promoting wound healing. A use of the composition according to any one of claims 1 to 4 for skin repair. The use of the composition as described in claim 9, wherein the skin is sensitive skin. A use of the composition according to any one of claims 1 to 4 for promoting hair growth.