Method for promoting exosome secretion of animal cells by using rose-derived exosomes

Abstract

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

Description

Technical Field

[0001] The present invention relates to a method for promoting exosome secretion in animal cells using rose-derived exosomes.

[0002] Furthermore, the present invention relates to a culture medium for promoting exosome secretion in animal cells, comprising rose-derived exosomes. Background Art

[0003] Recently, it has been reported that the cellular secretome contains a variety of bioactive molecules that regulate cell behavior. In particular, the cellular secretome contains "exosomes" or "extracellular vesicles" that have intercellular signaling functions, and therefore their components and functions have been actively studied.

[0004] Cells shed a variety of membrane vesicles into their extracellular environment, and these released vesicles are generally referred to as extracellular vesicles (EVs). EVs are also referred to as cell membrane-derived vesicles, extranuclear granules, shedding vesicles, microparticles, exosomes, etc., and in some cases are used to distinguish them from exosomes. Depending on the isolation environment, conditions, and methods, extracellular vesicles can have the same meaning as exosomes and can also refer to nanovesicles that have the same or similar size as exosomes but have a composition different from that of exosomes.

[0005] Exosomes are vesicles with a size of tens to hundreds of nanometers, which contain a phospholipid bilayer membrane with a structure similar to that of a cell membrane. Such exosomes contain proteins, nucleic acids (mRNA, miRNA, etc.), etc., which are called exosome cargo. It is known that exosome cargo includes a wide range of signal transduction factors, and these signal transduction factors are specific to cell types and are regulated differently depending on the environment of the secreting cell. It is known that exosomes are intercellular signal transduction mediators secreted by cells, and the various cell signals transmitted by them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis and necrosis of target cells. Exosomes contain specific genetic material and bioactive factors, which depend on the nature and state of the cells from which the exosomes are derived. Exosomes derived from proliferating stem cells regulate cell behavior, such as cell migration, proliferation and differentiation, and reproduce the characteristics of stem cells involved in tissue regeneration (Nature Review Immunology 2002(2) 569-579).

[0006] In other words, exosomes, known as cellular "avatars," contain bioactive factors, such as growth factors, similar to cells, and serve as vehicles for transferring these bioactive factors between cells, that is, mediating intercellular communication. Exosomes are known to be released not only from animal cells (e.g., stem cells, immune cells, fibroblasts, skin cells, and cancer cells), but also from cells of various organisms (e.g., plants, bacteria, fungi, and algae). For example, exosomes can be isolated from conditioned medium from plant cells, conditioned medium from plant callus, conditioned medium from plant stem cells, plant juices, or equivalent plant biological solutions, as well as conditioned medium from stem cells, immune cells, fibroblasts, skin cells, cancer cells, and the like.

[0007] Roses are cultivated in a wide range of regions, including the boreal, subarctic, temperate, and subtropical zones of the Northern Hemisphere, and their extracts are used in perfumes and cosmetics. Rose stem cell-derived exosomes are known to exhibit skin regeneration, improved skin elasticity, and wrinkle reduction (Korean Patent Publication No. 10-2058444), anti-inflammatory effects (Korean Patent Publication No. 10-2341932), and skin whitening effects (Korean Patent Publication No. 10-2261434). However, further detailed research on the characteristics and functions of rose-derived exosomes is needed.

[0008] The present inventors conducted intensive studies on rose-derived exosomes, and thus completed the present invention by finding that treating animal cells with rose-derived exosomes during their culture promotes exosome secretion from the animal cells.

[0009] At the same time, it should be understood that the content described as background technology is only intended to help understand the background of the present invention and is not considered to be prior art for the present invention. Summary of the Invention

[0010] An object of the present invention is to provide a method for promoting exosome secretion in animal cells using rose-derived exosomes.

[0011] Another object of the present invention is to provide a culture medium for promoting exosome secretion in animal cells, which comprises rose-derived exosomes.

[0012] However, the objects of the present invention as described above are illustrative, and the scope of the present invention is not limited thereby. In addition, other objects and advantages of the present invention will be more apparent from the following description, the appended claims and the accompanying drawings. DETAILED DESCRIPTION

[0013] As used herein, the term "rose (Rosa spp.)" refers to plants belonging to the genus Rosa, class Dicotyledons, order Rosales, family Rosaceae, and includes all wild species and cultivated garden species.

[0014] As used herein, the term "exosome" refers to vesicles measuring tens to hundreds of nanometers in size (preferably approximately 30 to 200 nm) that contain a phospholipid bilayer membrane with a structure similar to that of a cell membrane. (However, the particle size of exosomes varies, depending on the type of cell from which they 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 other substances, known as exosomal cargo. Exosomal cargo is known to include a wide range of signaling factors, and these signaling factors are cell-type specific and are regulated differently depending on the environment of the secreting cell. Exosomes are known to be intercellular signaling mediators secreted by cells, and various cell signals transmitted therethrough regulate cell behaviors, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells.

[0015] In the case of plants such as roses, "exosomes" refer to nanosized vesicles secreted or released into the extracellular space by plant cells and having a membrane structure, also known as extracellular vesicles, exosome-like vesicles, or exosome-like particles.

[0016] Furthermore, in the case of animal cells, “exosomes” include not only extracellular vesicles having a phospholipid bilayer membrane structure and specific markers that are secreted by animal cells and released into the extracellular space, but also vesicles having a nano-sized vesicle structure with a composition similar to that of exosomes (e.g., exosome-like vesicles or exosome-like particles).

[0017] As used herein, the term "exosome" used in connection with the promotion of exosome secretion is used in the sense of encompassing extracellular vesicles.

[0018] As used herein, the term "rose-derived exosomes" is intended to include all exosomes isolated from, for example, conditioned medium of rose plant cells, conditioned medium of rose callus tissue, conditioned medium of rose plant stem cells, rose juice, or rose biological solutions equivalent thereto, or all exosomes derived from (e.g., secreted or released from) rose plant cells or rose plant stem cells.

[0019] In the present invention, the type of animal cells from which exosomes are secreted is not limited, but as examples that do not limit the scope of the invention, they can be stem cells, immune cells, or skin cells. Stem cells can 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 can be T cells, B cells, NK cells, cytotoxic T cells, dendritic cells, or macrophages. Skin cells can be keratinocytes or dermal fibroblasts.

[0020] As an example that does not limit the scope of the present invention, adult stem cells can be at least one type of adult stem cells selected from the following: mesenchymal stem cells, human mesenchymal stromal cells, human mesenchymal stem cells, and multipotent stem cells. Mesenchymal stem cells can be mesenchymal stem cells derived from at least one tissue selected from the following: umbilical cord, umbilical cord blood, bone marrow, adipose tissue, muscle, nerve, skin, amnion, Wharton's jelly, and placenta. Preferably, the adult stem cells can be mesenchymal stem cells, such as adipose-derived, bone marrow-derived, umbilical cord-derived, or umbilical cord blood-derived stem cells, more preferably adipose-derived stem cells, and even more preferably human adipose-derived stem cells. The type of stem cells, immune cells, or skin cells is not limited, as long as they do not pose a risk of pathogen infection and do not cause immune rejection, but can preferably be human stem cells, human immune cells, or human skin cells.

[0021] However, various animal cells currently used in the art or that may be used in the future can be used, as long as they do not cause adverse effects on the human body. For example, HEK293 cells or HEK293T cells can also be used. Therefore, it should be understood that the human adipose-derived stem cells used in the examples described later are an example of animal cells that can be used in the present invention, and the present invention is not limited thereto.

[0022] The present invention provides a method for promoting exosome secretion in animal cells, comprising treating the animal cells with rose-derived exosomes.

[0023] In the method for promoting exosome secretion of animal cells according to one embodiment of the present invention, treating the animal cells with rose-derived exosomes may include adding rose-derived exosomes to a culture medium of the animal cells and culturing the animal cells in the culture medium.

[0024] In the method for promoting exosome secretion from animal cells according to one embodiment of the present invention, the exosomes secreted by animal cells can be quantified by the exosome content (tetraspanin content, such as CD81 content) per animal cell (or animal cell conditioned medium) or the number of exosome particles per animal cell (or animal cell conditioned medium).

[0025] The present invention provides a culture medium for promoting exosome secretion in animal cells, which comprises rose-derived exosomes.

[0026] Beneficial effects

[0027] According to the present invention, treating animal cells with rose-derived exosomes can promote exosome secretion in animal cells.

[0028] It should be understood that the scope of the present invention is not limited to the above-mentioned effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Graph showing particle size distribution and particle counts obtained by nanoparticle tracking analysis (NTA) of a culture supernatant obtained after culturing human adipose-derived stem cells in a medium not containing rose-derived exosomes (hereinafter referred to as “untreated control” or “control”).

[0030] Figure 2 is a graph showing the particle size distribution and particle number obtained by performing nanoparticle tracking analysis (NTA) on the culture supernatant obtained after culturing human adipose-derived stem cells in a medium supplemented with a low concentration of rose-derived exosomes.

[0031] Figure 3 Graph showing particle size distribution and particle counts obtained by nanoparticle tracking analysis (NTA) of a culture supernatant obtained after culturing human adipose-derived stem cells in a medium supplemented with a medium concentration of rose-derived exosomes.

[0032] Figure 4 Graph showing particle size distribution and particle counts obtained by nanoparticle tracking analysis (NTA) of a culture supernatant obtained after culturing human adipose-derived stem cells in a medium supplemented with a high concentration of rose-derived exosomes.

[0033] Figure 5 is a comparative graph showing that when human adipose-derived stem cells were cultured in a medium containing rose-derived exosomes according to one embodiment of the present invention, the number of particles per mL of conditioned medium (i.e., the amount of exosome secretion) was significantly increased compared to an untreated control.

[0034] Figure 6 is a comparative graph showing that when human adipose-derived stem cells are cultured in a medium containing rose-derived exosomes according to one embodiment of the present invention, the exosome content (the content of the exosome marker CD81) per mL of conditioned medium is increased compared to an untreated control.

[0035] Example

[0036] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the present invention and are not intended to limit or restrict the scope of the present invention. Those skilled in the art can easily infer from the detailed description and examples of the present invention, which are interpreted as falling within the scope of the present invention. The references mentioned in the present invention are incorporated herein by reference.

[0037] It should be understood that throughout this specification, when any part is referred to as “comprising” any component, unless otherwise specified, it does not exclude other components but may further comprise other components.

[0038] Example 1: Preparation of rose callus and preparation of rose-derived exosomes

[0039] Callus is induced from rose petals, leaves, stems, roots, and / or embryos, and cells of the induced rose callus are cultured according to plant callus preparation and culture methods known in the art. Well-growing callus is then selected and cultured in large quantities to prepare conditioned medium from the rose callus. The conditioned medium from the rose callus is filtered through a 0.22 μm filter to remove impurities such as cell debris, waste products, and large particles. Tangential flow filtration (TFF) is used to separate rose-derived exosomes from the filtered conditioned medium.

[0040] Example 2: Cell culture

[0041] Human adipose-derived stem cells were suspended in DMEM medium containing 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 μg / mL streptomycin. They were then seeded into flasks and cultured in an incubator at 37°C under 5% CO2. For the low-, medium-, and high-concentration treatment groups of rose-derived exosomes, rose-derived exosomes were added at a final concentration of 2.5×10 8 particles / mL ( Figure 2 、 Figure 5 and Figure 6 (shown as "L"), 8.0×108 particles / mL ( Figure 3 、 Figure 5 and Figure 6 denoted as "M"), and 2.5×10 9 particles / mL ( Figure 4 、 Figure 5 and Figure 6 was added to the culture medium.

[0042] When the cells reached 80% or higher confluence, 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 penicillin and 100 μg / mL streptomycin.

[0043] Afterwards, the untreated control group ( Figure 5 and Figure 6 Human adipose-derived stem cells (HSCs) were cultured for 24 to 72 hours in groups treated with low, medium, and high concentrations of rose-derived exosomes (shown as "control"), as well as in groups treated with low, medium, and high concentrations of rose-derived exosomes. Culture supernatants were collected from each group. Following supernatant collection, cell counts were measured using a cell counter in the untreated control group and the groups treated with low, medium, and high concentrations of rose-derived exosomes.

[0044] Example 3: Analysis of exosome particle size and distribution and evaluation of exosome secretion

[0045] For each supernatant collected from the untreated control group and the low-, medium-, and high-concentration rose-derived exosome-treated groups in Example 2, the particle size and concentration were measured by nanoparticle tracking analysis (NTA) using ZetaView (purchased from Particle Metrix). The measured NTA results were Figures 1 to 4 It was determined that the number of exosome particles in the collected supernatant increased by 36% in the low-concentration rose-derived exosome-treated group, 47% in the medium-concentration rose-derived exosome-treated group, and 178% in the high-concentration rose-derived exosome-treated group compared to the untreated control ( Figure 5 ).

[0046] Therefore, it was determined that when human adipose-derived stem cells were cultured in a culture medium containing rose-derived exosomes according to one embodiment of the present invention, that is, when human adipose-derived stem cells were treated with rose-derived exosomes, the number of exosome particles (that is, the amount of exosome secretion) was significantly increased compared to the untreated control.

[0047] Example 4: Evaluation of exosome secretion using exosome-specific markers

[0048] Since CD81 is a representative positive marker for exosomes, CD81 levels are linearly proportional to exosome levels. An increase in CD81 levels indicates a proportional increase in exosome levels. Based on this principle, the following analysis analyzed the exosome content (i.e., exosome secretion) in the supernatants collected from the untreated control group and the groups treated with low, medium, and high concentrations of rose-derived exosomes in Example 2. For this purpose, an exosome-human CD81 flow cytometry reagent was used. Each supernatant was mixed with the exosome-human CD81 flow cytometry reagent overnight, and then each mixture was reacted with PE mouse anti-human CD81 for one hour. Following the reaction, the PE mean fluorescence intensity (MFI) of the samples was measured using flow cytometry.

[0049] Concurrently, linear regression analysis was performed using the mean fluorescence intensity of PE to calculate CD81 content. Specifically, linear regression analysis was performed using the concentrations of serially diluted CD81 protein and the corresponding MFI values. A standard quantitative analysis plot generated by linear regression analysis was used to determine the exosome content (CD81 content) in each supernatant.

[0050] like Figure 6 As shown, comparison of CD81 content confirmed that when human adipose-derived stem cells were cultured in a medium containing rose-derived exosomes (ie, treated with rose-derived exosomes), the exosome content (CD81 content) in the supernatant was increased compared to an untreated control.

[0051] Therefore, it was confirmed again that when human adipose-derived stem cells were cultured in a culture medium containing rose-derived exosomes according to one embodiment of the present invention, that is, when human adipose-derived stem cells were treated with rose-derived exosomes, the amount of exosome secretion increased.

[0052] Although the present invention has been described with reference to some embodiments, the scope of the present invention is not limited to these embodiments. It will be understood by any person skilled in the art that various modifications and changes can be made without departing from the spirit and scope of the present invention, and these modifications and changes also fall within the scope of the present invention.

Claims

1. A method for promoting exosome secretion in animal cells, comprising treating the animal cells with rose-derived exosomes. 2 . The method of claim 1 , wherein treating the animal cells with rose-derived exosomes comprises adding the rose-derived exosomes to a culture medium of the animal cells, and culturing the animal cells in the culture medium.

3. The method of claim 1 or 2, wherein the rose-derived exosomes are isolated from conditioned medium of rose plant cells, conditioned medium of rose callus, conditioned medium of rose plant stem cells, rose juice, or a rose biological solution equivalent thereto, or are derived from rose plant cells or rose plant stem cells.

4. A culture medium for promoting exosome secretion in animal cells, comprising rose-derived exosomes.

5. The culture medium of claim 4, wherein the rose-derived exosomes are isolated from a conditioned medium of rose plant cells, a conditioned medium of rose callus tissue, a conditioned medium of rose plant stem cells, rose juice, or a rose biological solution equivalent thereto, or are derived from rose plant cells or rose plant stem cells.

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