MÉTODO PARA PROMOVER A SECREÇÃO DE EXOSSOMAS DE CÉLULAS DE ANIMAIS COM O USO DE EXOSSOMAS DERIVADOS DE ROSAS

Abstract

The present invention provides a method for promoting the secretion of exosomes from animal cells, comprising a step of treating animal cells with rose-derived exosomes. According to the present invention, the secretion of exosomes from animal cells can be promoted by treating animal cells with rose-derived exosomes.

Description

1 / 14 “METHOD FOR PROMOTING THE SECRETION OF EXOSOMES FROM ANIMAL CELLS USING ROSE-DERIVED EXOSOMES FIELD OF THE TECHNIQUE

[001] The present invention relates to a method for promoting the secretion of exosomes from animal cells using exosomes derived from roses.

[002] Furthermore, the present invention relates to a means for promoting the secretion of exosomes from animal cells, comprising exosomes derived from roses. PREVIOUS TECHNIQUE

[003] Recently, there have been reports that cell secretomes contain various bioactive molecules that regulate cell behavior. In particular, cell secretomes contain exosomes or extracellular vesicles that have intercellular signaling functions, and therefore studies on their components and functions have been actively conducted.

[004] Cells release various membranous vesicles into their extracellular environment, and these released vesicles are generally called extracellular vesicles (EVs). EVs are also referred to as cell membrane-derived vesicles, ectosomes, release vesicles, microparticles, exosomes, etc., and are also used interchangeably with exosomes in some cases. Depending on the environment, conditions, and isolation methods, extracellular vesicles may have the same meaning as exosomes and may also include nanovesicles that are the same size or similar in size to exosomes but have a different composition than exosomes.

[005] The exosome is a vesicle tens to hundreds of nanometers in size, comprising a bilayer membrane Petition 870250081092, dated 09 / 09 / 2025, pages 80 / 102 2 / 14 phospholipid with the same structure as the cell membrane. This exosome contains proteins, nucleic acids (mRNA, miRNA, etc.) and similar substances that are called the exosome cargo. The exosome cargo is known to include a wide range of signaling factors, and these signaling factors are specific to cell types and regulated differently depending on the environment of the secreting cells. The exosome is known to be a mediator of intercellular signaling secreted by cells, and various cellular signals transmitted by it regulate cellular behaviors, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. The exosome contains specific genetic materials and bioactive factors, depending on the nature and state of the cells from which the exosome was derived.The exosome derived from proliferating stem cells regulates cell behavior, such as migration, proliferation and cell differentiation, and recapitulates the characteristics of stem cells involved in tissue regeneration (Nature Review Immunology 2002 (2) 569579).

[006] In other words, exosomes, called cell avatars, contain bioactive factors such as growth factors, similar to cells, and serve as carriers that transmit bioactive factors between cells, that is, they serve to mediate cell-to-cell communication. Exosomes are known to be released not only from animal cells, such as stem cells, immune cells, fibroblasts, skin cells, and cancer cells, but also from cells of various organisms, such as plants, bacteria, fungi, and algae. For example, exosomes can be isolated from conditioned media Petition 870250081092, dated 09 / 09 / 2025, pages 81 / 102 3 / 14 of plant cells, conditioned media of plant calluses, conditioned media of plant stem cells, plant juice or a biological solution of a plant equivalent to them, as well as conditioned media of stem cells, immune cells, fibroblasts, skin cells, cancer cells or the like.

[007] Meanwhile, roses are cultivated in a wide range of areas, such as cold, subarctic, temperate, and subtropical zones of the northern hemisphere, and their extracts are used in perfumes and cosmetics. Exosomes derived from rose stem cells are known to exhibit efficacy in skin regeneration, improved skin elasticity, and wrinkle reduction (Patent Publication No. KR 10-2058444), anti-inflammatory efficacy (Patent Publication No. KR 10-2341932), and skin-brightening efficacy (Patent Publication No. KR 10-2261434). However, more detailed research is needed on the characteristics and functions of rose-derived exosomes.

[008] The present inventor conducted intensive research on rose-derived exosomes and, as a result, completed the present invention by confirming that treatment of animal cells with rose-derived exosomes during animal cell culture promotes the secretion of exosomes from animal cells.

[009] Meanwhile, it should be understood that the materials described as the background of the art are intended only to assist in understanding the background of the present invention and are not admitted as prior art against the present invention. SUMMARY OF THE INVENTION Petition 870250081092, dated 09 / 09 / 2025, pages 82 / 102 4 / 14

[010] One objective of the present invention is to provide a method for promoting the secretion of exosomes from animal cells using rose-derived exosomes.

[011] Another objective of the present invention is to provide a means for promoting the secretion of exosomes from animal cells, comprising exosomes derived from roses.

[012] However, the objects of the present invention, as described above, are illustrative and the scope of the present invention is not limited thereto. Furthermore, other objects and advantages of the present invention will become more apparent from the following description, the appended claims and the accompanying drawings. DETAILED DESCRIPTION OF THE INVENTION

[013] As used in this document, the term rose (Rosa spp.) refers to plants belonging to the genus Rosa, in the family Rosaceae, to the order Rosales and to the class Dicotyledoneae, and includes all wild species and cultivated garden species.

[014] As used in this document, the term exosomes refers to vesicles tens to hundreds of nanometers in size (preferably about 30 to 200 nm), comprising a phospholipid bilayer membrane with the same structure as that of the cell membrane (however, the size of exosome particles varies depending on the type of cell from which the exosomes are isolated, the isolation method, and the measurement method) (Vasiliy S. Chernyshev et al., Size and shape characterization of hydrated and desiccated exosomes, Anal Bioanal Chem, (2015) DOI 10.1007 / s00216-015-8535-3). These exosomes contain proteins, nucleic acids (mRNA, miRNA, etc.) and the like, which are Petition 870250081092, dated 09 / 09 / 2025, pages 83 / 102 5 / 14 called exosome payload. The exosome payload is known to include a wide range of signaling factors, and these signaling factors are specific to cell types and regulated differently depending on the environment of the secretory cells. Exosomes are known to be mediators of intercellular signaling secreted by cells, and various cellular signals transmitted by them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells.

[015] In the case of plants such as roses, exosomes refer to nanometer-sized vesicles secreted or released from plant cells into extracellular spaces and with a membrane structure, also called extracellular vesicles, exosome-like vesicles or exosome-like particles.

[016] Furthermore, in the case of animal cells, exosomes include not only extracellular vesicles secreted by animal cells and released into the extracellular space, with a phospholipid bilayer membrane structure and specific markers, but also vesicle-like structures of nanometer size with a composition similar to exosomes (e.g., exosome-like vesicles or exosome-like particles).

[017] As used in this document, the term exosome used in relation to promoting exosome secretion is used in a sense that encompasses extracellular vesicles.

[018] As used in this document, the term rose-derived exosomes includes all exosomes Petition 870250081092, dated 09 / 09 / 2025, pages 84 / 102 6 / 14 isolates from, for example, a conditioned medium of rose plant cells, a conditioned medium of rose callus, a conditioned medium of rose plant stem cells, rose juice or a rose biological solution equivalent to it, or derived (e.g., secreted or released) from rose plant cells or rose plant stem cells.

[019] In the present invention, the type of animal cells from which exosomes are secreted is not limited, but as an example that does not limit the scope of the present invention, they may be stem cells, immune cells, or skin cells. Stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stem cells derived from embryonic stem cells, or mesenchymal stem cells derived from induced pluripotent stem cells. Immune cells may be T cells, B cells, NK cells, cytotoxic T cells, dendritic cells, or macrophages. Skin cells may be keratinocytes or dermal fibroblasts.

[020] As an example that does not limit the scope of the present invention, adult stem cells may be at least one type of adult stem cells selected from the group consisting of mesenchymal stem cells, human-derived mesenchymal stromal cells, human-derived mesenchymal stem cells, and multipotent stem cells. Mesenchymal stem cells may be mesenchymal stem cells derived from at least one tissue selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, adipose tissue, muscle, nerve, skin, amniotic membrane, Wharton's jelly, and placenta. Petition 870250081092, dated 09 / 09 / 2025, pages 85 / 102 7 / 14 Preferably, adult stem cells may be mesenchymal stem cells, for example, stem cells derived from adipose tissue, bone marrow, umbilical cord or umbilical cord blood, more preferably stem cells derived from adipose tissue and, even more preferably, stem cells derived from human adipose tissue. The type of stem cells, immune cells or skin cells is not limited, provided they do not pose a risk of infection by pathogens and do not cause immune rejection, but may preferably be human-derived stem cells, human-derived immune cells or human-derived skin cells.

[021] However, it is clear that it is possible to use various animal cells that are used in the technique or may be used in the future, provided they do not cause adverse effects on the human body. For example, it is also possible to use HEK293 cells or HEK293T cells. Thus, it should be understood that the stem cell derived from human adipose tissue used in the examples described below is an example of animal cells that can be used in the present invention, and the present invention is not limited to this.

[022] The present invention provides a method for promoting the secretion of exosomes from animal cells, comprising treating animal cells with rose-derived exosomes.

[023] In the method for promoting the secretion of exosomes from animal cells according to one embodiment of the present invention, the treatment of animal cells with rose-derived exosomes may include adding the rose-derived exosomes to an animal cell culture medium. Petition 870250081092, dated 09 / 09 / 2025, pages 86 / 102 8 / 14 and the culture of animal cells in culture medium.

[024] In the method for promoting the secretion of exosomes from animal cells according to an embodiment of the present invention, the exosomes secreted by animal cells can be quantified by the exosome content (tetraspanin content, for example, CD81 content) per animal cell (or conditioned medium per animal cell) or the number of exosome particles per animal cell (or conditioned medium per animal cell).

[025] The present invention provides a means for promoting the secretion of exosomes from animal cells, comprising exosomes derived from roses. ADVANTAGEOUS EFFECTS

[026] According to the present invention, treatment of animal cells with rose-derived exosomes can promote the secretion of exosomes from animal cells.

[027] It should be understood that the scope of the present invention is not limited to the effects mentioned above. BRIEF DESCRIPTION OF THE DRAWINGS

[028] Figure 1 is a graph showing the particle size distribution and particle number obtained by performing nanoparticle tracking analysis (NTA) of a culture supernatant obtained after culturing human adipose tissue-derived stem cells in a medium that does not contain rose-derived exosomes (hereinafter referred to as the untreated control or control).

[029] Figure 2 is a graph showing the particle size distribution and particle number obtained by performing nanoparticle tracking analysis (NTA) of a culture supernatant obtained after cell culture. Petition 870250081092, dated 09 / 09 / 2025, pages 87 / 102 9 / 14 stem cells derived from human adipose tissue in a medium supplemented with a low concentration of rose-derived exosomes.

[030] Figure 3 is a graph showing the particle size distribution and particle number obtained by performing nanoparticle tracking analysis (NTA) of a culture supernatant obtained after culturing human adipose tissue-derived stem cells in a medium supplemented with a moderate concentration of rose-derived exosomes.

[031] Figure 4 is a graph showing the particle size distribution and number of particles obtained by performing nanoparticle tracking analysis (NTA) of a culture supernatant obtained after culturing human adipose tissue-derived stem cells in a medium supplemented with a high concentration of rose-derived exosomes.

[032] Figure 5 is a comparative graph showing that the number of particles per ml of conditioned medium (i.e., the amount of exosome secretion) increased markedly compared with the untreated control when stem cells derived from human adipose tissue were cultured in a medium containing rose-derived exosomes according to an embodiment of the present invention.

[033] Figure 6 is a comparative graph showing that the exosome content (CD81 content, which is an exosome marker) per ml of conditioned medium increased compared to the untreated control when human adipose tissue-derived stem cells were cultured in a medium containing rose-derived exosomes according to a form of Petition 870250081092, dated 09 / 09 / 2025, pages 88 / 102 10 / 14 implementation of the present invention. EXAMPLES

[034] Hereafter in this document, the present invention will be described in more detail with reference to the following examples. However, the following examples are only to illustrate the present invention and are not intended to limit or restrict the scope of the present invention. Those that can be readily inferred by persons skilled in the art from the detailed description and examples of the present invention are interpreted as belonging to the scope of the present invention. The references referred to in the present invention are incorporated herein in their entirety by way of reference.

[035] Throughout this specification, it should be understood that when a part is referred to as comprising any component, it does not exclude other components, but may include other components unless otherwise indicated. EXAMPLE 1: PREPARATION OF ROSE CALLUS AND ROSE-DERIVED EXOSOMES

[036] According to known plant callus preparation and cultivation methods in the art, calluses were induced from rose petals, leaves, stems, roots, and / or embryos, and the induced rose callus cells were cultured. Then, a callus with a good growth state was selected and cultivated in large quantities to prepare rose callus conditioned medium. The rose callus conditioned medium was filtered through a 0.22 μm filter to remove impurities such as cellular debris, residues, and large particles. Rose-derived exosomes were isolated from the filtered conditioned medium using a Petition 870250081092, dated 09 / 09 / 2025, pages 89 / 102 11 / 14 tangential flow filtration (TFF) method. EXAMPLE 2: CELL CULTURE

[037] Stem cells derived from human adipose tissue were suspended in DMEM culture medium containing 10% fetal bovine serum (FBS), 100 units / ml of penicillin, and 100 μg / ml of streptomycin. They were then inoculated into a flask and cultured in an incubator at 37 °C under 5% CO2. For the low, moderate, and high concentration treatment groups of rose-derived exosomes, rose-derived exosomes were added to the culture medium at final concentrations of 2.5*108 particles / ml (indicated as L in Figures 2, 5, and 6), 8.0*108 particles / ml (indicated as M in Figures 3, 5, and 6), and 2.5*109 particles / ml (indicated as H in Figures 4, 5, and 6), respectively.

[038] When the cells reached a confluence of 80% or more, the cells were washed with phosphate-buffered saline (PBS; purchased from Thermo Scientific) and then the culture medium was replaced with DMEM medium containing 100 units / ml of penicillin and 100 pg / ml of streptomycin.

[039] Subsequently, stem cells derived from human adipose tissue were cultured for 24 to 72 hours in the untreated control group (indicated as Control in Figures 5 and 6) and in the treatment groups with rose-derived exosomes of low, moderate, and high concentration, respectively. The supernatant of the culture medium from each group was collected. After the supernatant collection was complete, the number of cells in the untreated control group and in the treatment groups with rose-derived exosomes of low, moderate, and high concentration were measured using a cell counter. Petition 870250081092, dated 09 / 09 / 2025, pp. 90 / 102 12 / 14 respectively. EXAMPLE 3: ANALYSIS OF EXOSOME PARTICLE SIZE AND DISTRIBUTION AND EVALUATION OF EXOSOME SECRETION QUANTITY

[040] For each of the supernatants collected from the untreated control group and the low-concentration, moderate-concentration, and high-concentration rose-derived exosome treatment groups in Example 2, particle size and concentration were measured by performing nanoparticle tracking analyses (NTA) using ZetaView (acquired from Particle Metrix). The measured NTA results are shown in Figures 1 to 4. It was confirmed that the number of exosome particles in the collected supernatant increased by 36% in the low-concentration rose-derived exosome treatment group, 47% in the moderate-concentration rose-derived exosome treatment group, and 178% in the high-concentration rose-derived exosome treatment group, respectively, compared to the untreated control (Figure 5).

[041] Therefore, it was confirmed that when stem cells derived from human adipose tissue were cultured in a medium containing rose-derived exosomes according to an embodiment of the present invention, i.e., when stem cells derived from human adipose tissue were treated with rose-derived exosomes, the number of exosome particles (i.e., the amount of exosome secretion) increased markedly compared with the untreated control. EXAMPLE 4: EVALUATION OF THE AMOUNT OF EXOSOME SECRETION WITH THE USE OF A SPECIFIC EXOSOME MARKER

[042] Since CD81 is a positive marker representative of exosomes, the CD81 content and the exosome content are Petition 870250081092, dated 09 / 09 / 2025, pages 91 / 102 13 / 14 linearly proportional to each other. An increase in CD81 content indicates a proportional increase in exosome content. Hereafter in this document, according to this principle, the exosome content (i.e., the amount of exosome secretion) was analyzed for each of the supernatants from the untreated control group and the treatment groups with low-concentration, moderate-concentration, and high-concentration rose-derived exosomes collected in Example 2. For this, the CD81 Exosome-Human Flow Detection Reagent was used. Each supernatant was mixed with CD81 exosome-human flow detection reagent overnight, and then each mixture reacted with mouse anti-human CD81 PE for 1 hour. After the reaction, the mean fluorescence intensity (MFI) of PE samples was measured by flow cytometry.

[043] Meanwhile, linear regression analysis was performed to calculate the CD81 content using the mean fluorescence intensity of the PE. That is, linear regression analysis was performed using serially diluted CD81 protein concentrations and the corresponding IMF values. The exosome content (CD81 content) in each supernatant was determined using the standard quantitative analysis plot generated from the linear regression analysis.

[044] As shown in Figure 6, a comparison of CD81 content confirmed that when human adipose tissue-derived stem cells were cultured in a medium containing rose-derived exosomes (i.e., treated with rose-derived exosomes), the exosome content (CD81 content) in the supernatant increased compared to the untreated control.

[045] Therefore, it was reconfirmed that when human adipose tissue-derived stem cells were cultured in Petition 870250081092, dated 09 / 09 / 2025, pages 92 / 102 14 / 14 a medium containing rose-derived exosomes according to one embodiment of the present invention, that is, when stem cells derived from human adipose tissue were treated with rose-derived exosomes, the amount of exosome secretion increased.

[046] Although the present invention has been described with reference to embodiments, the scope of the present invention is not limited to those embodiments. Anyone skilled in the art will observe that various modifications and changes are possible without departing from the spirit and scope of the present invention, and such modifications and changes also fall within the scope of the present invention. Petition 870250081092, dated 09 / 09 / 2025, pages 93 / 102

Claims

1 / 1 CLAIMS 1. Method for promoting the secretion of exosomes from animal cells characterized by comprising the treatment of animal cells with rose-derived exosomes.

2. Method according to claim 1, characterized by treating animal cells with rose-derived exosomes comprising adding the rose-derived exosomes to an animal cell culture medium and culturing the animal cells in the culture medium.

3. A method according to claim 1 or 2, characterized in that the rose-derived exosomes are isolated from a conditioned medium of rose plant cells, a conditioned medium of rose calli, a conditioned medium of rose plant stem cells, rose juice or a rose biological solution equivalent thereto, or derived from rose plant cells or rose plant stem cells.

4. A medium for promoting the secretion of exosomes from animal cells, characterized by comprising rose-derived exosomes.

5. Medium, according to claim 4, characterized in that the rose-derived exosomes are isolated from a conditioned medium of rose plant cells, a conditioned medium of rose calli, a conditioned medium of rose plant stem cells, rose juice or a rose biological solution equivalent thereto, or derived from rose plant cells or rose plant stem cells. Petition 870250081092, dated 09 / 09 / 2025, pp. 94 / 102

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