A method to promote exosome secretion from animal cells using rose-derived exosomes.

Abstract

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

Description

Technical Field

[0001] The present invention relates to a method for promoting the secretion of exosomes from animal cells by using exosomes derived from roses.

[0002] The present invention also relates to a culture medium for promoting exosome secretion of animal cells, which contains exosomes derived from roses.

Background Art

[0003] Recently, studies have been reported that cell secretions (secretome) contain various bioactive factors that regulate cell behavior. In particular, cell secretions contain "exosomes" or "extracellular vesicles" that have an intercellular signaling function, and research on their components and functions has been actively carried out.

[0004] Cells release various membrane-type vesicles into the extracellular environment, and usually, such released vesicles are called extracellular vesicles (EVs). Extracellular vesicles are called vesicles derived from the cell membrane, ectosomes, shedding vesicles, microparticles, exosomes, etc., and in some cases, they may be used separately from exosomes. Depending on the separation environment, conditions, methods, etc., extracellular vesicles may have the same meaning as exosomes, and may have the same or similar size as exosomes, but may also include nanovesicles that do not have the composition of exosomes.

[0005] Exosomes are endoplasmic reticulum (ER) structures measuring tens to hundreds of nanometers in size, possessing a biphospholipid membrane identical to that of the cell membrane. They contain exosome cargo, which includes proteins and nucleic acids (mRNA, miRNA, etc.). Exosome cargo contains a wide range of signaling factors, known to be cell-type specific and regulated differently depending on the environment of the secreting cell. Exosomes are intercellular signaling media secreted by cells, and the various cellular signals transmitted through them are known to regulate cellular behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Exosomes contain specific genetic material and bioactive factors depending on the nature and state of the cell from which they originate. In the case of exosomes derived from proliferating stem cells, they regulate cellular behavior such as cell migration, proliferation, and differentiation, reflecting the stem cell characteristics related to tissue regeneration (Non-Patent Literature 1).

[0006] In other words, exosomes, often called cellular avatars, contain bioactive factors such as growth factors, just like cells themselves. However, they act as messengers that transport these bioactive factors between cells, essentially playing a role in 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 the cells of various organisms, including plants, bacteria, fungi, and algae. For example, exosomes can be isolated not only from the culture media of stem cells, immune cells, fibroblasts, skin cells, and cancer cells, but also from plant cell culture media, plant callus culture media, plant stem cell culture media, plant juices, or equivalent plant biological solutions.

[0007] Meanwhile, roses are cultivated in a wide range of regions in the Northern Hemisphere, including the polar, subarctic, temperate, and subtropical zones, and their extracts are used in perfumes and cosmetics. Rose stem cell-derived exosomes are known to exhibit efficacy in skin regeneration, improvement of skin elasticity and wrinkle reduction (Patent Document 1), anti-inflammatory effects (Patent Document 2), and skin whitening effects (Patent Document 3). However, further detailed research is needed regarding the characteristics and functions of rose-derived exosomes.

[0008] The inventors of this invention have been diligently researching rose-derived exosomes and have confirmed that treating animal cells with rose-derived exosomes during culture promotes the secretion of exosomes from the animal cells, thereby completing the present invention.

[0009] On the other hand, it should be understood that the matters described above as background technology are merely for the purpose of advancing understanding of the background of the present invention, and are not cited as approval that they can be used as "prior art" of the present invention. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] Korean Registered Patent Publication No. 10-2058444 [Patent Document 2] Korean Registered Patent Publication No. 10-2341932 [Patent Document 3] Korean Registered Patent Publication No. 10-2261434 [Non-patent literature]

[0011] [Non-Patent Document 1] Nature Review Immunology 2002(2)569-579 [Non-Patent Document 2] 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 [Overview of the Initiative] [Problems that the invention aims to solve]

[0012] The object of the present invention is to provide a method for promoting the secretion of exosomes from animal cells using rose-derived exosomes.

[0013] Another object of the present invention is to provide a culture medium for promoting exosome secretion from animal cells, which contains rose-derived exosomes.

[0014] However, the problems of the present invention described above are illustrative and do not limit the scope of the invention. Furthermore, other objects and advantages of the present invention will become clearer from the detailed description of the invention below, the claims and drawings. [Means for solving the problem]

[0015] In this specification, the term "rose (Rosa spp.)" refers collectively to plants belonging to the dicotyledonous plant group, the order Rosales, the family Rosaceae, and the genus Rosa, and includes both wild species and cultivated varieties.

[0016] In this specification, the term "exosomes" refers to endoplasmic reticulum (ER) having a biphospholipid membrane with the same structure as the cell membrane, measuring tens to hundreds of nanometers (preferably about 30-200 nm) in size (however, the particle size of exosomes may vary depending on the type of cell to be isolated, the isolation method, and the measurement method) (Non-Patent Literature 2). Exosomes contain proteins and nucleic acids (mRNA, miRNA, etc.) called exosome cargo. Exosome cargo contains a wide range of signaling factors, and these signaling factors are known to be cell type specific and regulated differently depending on the environment of the secreting cell. Exosomes are intercellular signaling media secreted by cells, and the various cellular signals transmitted by them are known to regulate cellular behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells.

[0017] In plants like roses, "exosomes" refer to nano-sized vesicles with membrane structures secreted or released from plant cells into the extracellular space, and are also called extracellular vesicles, exosome-like vesicles, or exosome-like particles.

[0018] Furthermore, in the case of animal cells, "exosomes" include not only extracellular vesicles with a biphospholipid membrane structure and characteristic markers that are secreted from animal cells and released into the extracellular space, but also all vesicles that have a nano-sized vesicle structure and a composition similar to that of exosomes (e.g., exosome-like vesicles or exosome-like particles).

[0019] Preferably, as used herein in relation to exosome secretion promotion, the term exosome is used to encompass extracellular vesicles.

[0020] The term "exosome derived from rose" used in the specification of the present invention means, for example, any exosomes that are separated from a rose plant cell culture solution, a rose callus culture solution, a rose plant stem cell culture solution, a rose juice extract, or a biological solution of rose equivalent thereto, or that are derived from rose plant cells or rose plant stem cells, such as secreted and / or released exosomes.

[0021] In the present invention, the type of animal cells from which exosomes are secreted is not limited. As one non-limiting example of the present invention, they may be stem cells, immune cells or skin cells. The stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), somatic stem cells, mesenchymal stem cells derived from embryonic stem cells, or mesenchymal stem cells derived from induced pluripotent stem cells. The immune cells may be T cells, B cells, NK cells, cytotoxic T cells, dendritic cells or macrophages. The skin cells may be keratinocytes or skin fibroblasts.

[0022] As a non-limiting example of the present invention, the somatic stem cells may be one or more somatic stem cells selected from the group consisting of mesenchymal stem cells, human-derived mesenchymal stromal cells, human-derived mesenchymal stem cells, and pluripotent stem cells. The mesenchymal stem cells may be mesenchymal stem cells derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, adipose tissue, muscle, nerve, skin, amnion, Wharton's jelly and placenta. Preferably, the somatic stem cells may be mesenchymal stem cells, such as stem cells derived from adipose tissue, bone marrow, umbilical cord or umbilical cord blood, more preferably adipose-derived stem cells, and even more preferably human adipose-derived stem cells. The types of the stem cells, immune cells or skin cells are not limited as long as there is no risk of infection by pathogens and no immune rejection reaction occurs, but preferably they may be human-derived stem cells, human-derived immune cells or human-derived skin cells.

[0023] However, it goes without saying that various animal cells currently used or potentially used in this industry can be used, as long as they do not cause adverse effects on the human body. For example, HEK293 cells and HEK293T cells can also be used. Therefore, the human adipose-derived stem cells used in the examples described later should be understood as just one example of animal cells that can be used in this invention, and it should be made clear that this invention is not limited to this.

[0024] The present invention provides a method for promoting exosome secretion from animal cells, comprising treating animal cells with rose-derived exosomes.

[0025] In a method for promoting exosome secretion from animal cells according to one specific example of the present invention, treating the animal cells with rose-derived exosomes may involve adding the rose-derived exosomes to the culture medium of the animal cells during cultivation and then culturing the animal cells.

[0026] In a method for promoting exosome secretion from animal cells according to one specific example of the present invention, the exosomes secreted from the animal cells can be quantified by the exosome content per animal cell (or animal cell culture medium) (tetraspanin content, e.g., CD81 content), or by the number of exosome particles per animal cell (or animal cell culture medium).

[0027] This invention provides a culture medium for promoting exosome secretion from animal cells, which contains rose-derived exosomes. [Effects of the Invention]

[0028] According to the present invention, the secretion of exosomes from animal cells can be promoted by treating animal cells with rose-derived exosomes.

[0029] On the other hand, the scope of the present invention is not limited by the effects described above. [Brief explanation of the drawing]

[0030] [Figure 1] This graph shows the particle size distribution and number of particles obtained by performing nanoparticle tracking analysis (NTA) on the culture supernatant obtained after culturing human adipose-derived stem cells in a medium that does not contain rose-derived exosomes (hereinafter referred to as the "untreated control group" or "control group"). [Figure 2] This graph shows the particle size distribution and number of particles 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. [Figure 3] This graph shows the particle size distribution and number of particles 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 moderate concentration of rose-derived exosomes. [Figure 4] This graph shows the particle size distribution and number of particles 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 high concentration of rose-derived exosomes. [Figure 5] This is a comparative graph showing that when human adipose-derived stem cells were cultured in a medium containing rose-derived exosomes according to one specific example of the present invention, the number of particles per mL of culture medium (i.e., the amount of exosome secreted) increased significantly compared to the untreated control group. [Figure 6] This is a comparative graph showing that when human adipose-derived stem cells were cultured in a medium containing rose-derived exosomes according to one specific example of the present invention, the exosome content (CD81 content, which is the label of exosomes) per mL of culture medium increased compared to an untreated control group. [Modes for carrying out the invention]

[0031] The present invention will be described in more detail below with reference to the following examples. However, the following examples are merely illustrative of the content of the present invention and do not limit or restrict the scope of the rights of the present invention. Anything that can be easily inferred by analogy to a person ordinary to the art to which the present invention belongs from the detailed description and examples of the present invention shall be interpreted as falling within the scope of the rights of the present invention. The references cited in the present invention are incorporated into the present invention as references.

[0032] When a specification states that a part of it "includes" a certain component, unless otherwise stated, this means that it may include other components rather than excluding them. [Examples]

[0033] <Example 1: Production of rose callus and rose-derived exosomes> Using plant callus production and culture methods known in this industry, callus was induced from rose petals, leaves, stems, roots, and / or embryos, and the induced rose callus was cultured. Callus with good growth was selected and cultured in large quantities to prepare a rose callus culture medium. The rose callus culture medium was filtered through a 0.22 μm filter to remove impurities such as cell debris, waste products, and large particles. Rose-derived exosomes were separated from the filtered culture medium using a tangential flow filtration (TFF) system.

[0034] <Example 2: Cell Culture> Human adipose-derived stem cells were suspended in DMEM culture medium containing 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 μg / mL streptomycin, then inoculated into flasks and cultured at 37°C in a 5% CO2 incubator. In the cases of low, medium, and high concentration treatments with rose-derived exosomes, rose-derived exosomes were added to the culture medium, resulting in final concentrations of 2.5 × 10⁶, respectively. 8 particles / mL (indicated as "L" in Figures 2, 5, and 6), 8.0 × 10 8particles / mL (indicated as "M" in Figures 3, 5, and 6) and 2.5 × 10 9 The solution was added to a concentration of particles / mL (indicated as "H" in Figures 4, 5, and 6).

[0035] Once the cell density (confluency) reached 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 penicillin and 100 μg / mL streptomycin.

[0036] Subsequently, human adipose-derived stem cells were cultured for 24 to 72 hours in the untreated control group (indicated as "control group" in Figures 5 and 6), the low-concentration rose-derived exosome treatment group, the medium-concentration rose-derived exosome treatment group, and the high-concentration rose-derived exosome treatment group, respectively, and the supernatant of each culture medium was collected. After the collection of the supernatant was complete, the number of cells in the untreated control group, the low-concentration rose-derived exosome treatment group, the medium-concentration rose-derived exosome treatment group, and the high-concentration rose-derived exosome treatment group was measured using a cell counter.

[0037] <Example 3: Analysis of exosome particle size and distribution, and evaluation of exosome secretion amount> The particle size and concentration of the untreated control supernatant, the low-concentration rose-derived exosome-treated supernatant, the medium-concentration rose-derived exosome-treated supernatant, and the high-concentration rose-derived exosome-treated supernatant recovered in Example 2 were measured by nanoparticle tracking analysis (NTA) using Zetaview (purchased 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 recovered supernatant of the low-concentration rose-derived exosome-treated supernatant, the medium-concentration rose-derived exosome-treated supernatant, and the high-concentration rose-derived exosome-treated supernatant increased by 36% (low-concentration), 47% (medium-concentration), and 178% (high-concentration) compared to the untreated control group, respectively (Figure 5).

[0038] Therefore, when human adipose-derived stem cells were cultured in a medium containing rose-derived exosomes according to one specific example of the present invention, i.e., when human adipose-derived stem cells were treated with rose-derived exosomes, it was confirmed that the number of exosome particles (i.e., the amount of exosome secreted) increased significantly compared to the untreated control group.

[0039] <Example 4: Evaluation of exosome secretion using exosome-specific markers> Since CD81 is a representative positive marker for exosomes, the CD81 content and exosome content are linearly related, meaning that an increase in CD81 content corresponds to a proportional increase in exosome content. In the following, following this principle, the exosome content (exosome secretion amount) was analyzed for the untreated control supernatant, the low-concentration exosome-treated supernatant from roses, the medium-concentration exosome-treated supernatant from roses, and the high-concentration exosome-treated supernatant from roses, all recovered in Example 2. An exosome-human CD81 flow detection reagent was used for this analysis. Each culture supernatant was mixed with the exosome-human CD81 flow detection reagent overnight, and then reacted with PE mouse anti-human CD81 for 1 hour. After the reaction was complete, the mean fluorescence intensity (MFI) of the samples was measured using flow cytometry.

[0040] On the other hand, linear regression analysis was performed to calculate the CD81 content using the average fluorescence intensity of PE. Specifically, linear regression analysis was performed using the concentration of CD81 protein prepared by serial dilution and the MFI value corresponding to that concentration. Using the standard quantitative analysis graph determined by this method, the exosome content (CD81 content) in each culture supernatant was determined.

[0041] As illustrated in Figure 6, a comparison of CD81 content revealed that when human adipose-derived stem cells were cultured (i.e., pretreated) in a medium containing rose-derived exosomes, the exosome content (CD81 content) in the supernatant increased compared to the untreated control group.

[0042] Therefore, we have reconfirmed that when human adipose-derived stem cells are cultured in a medium containing rose-derived exosomes according to one specific example of the present invention, i.e., when human adipose-derived stem cells are treated with rose-derived exosomes, the amount of exosome secretion increases.

[0043] Although the present invention has been described above with reference to the embodiments described above, the present invention is not limited thereto. Those skilled in the art will understand that modifications and changes can be made without departing from the spirit and scope of the present invention, and that such modifications and changes also belong to the present invention.

Claims

1. A method for promoting exosome secretion from animal cells, including treating animal cells with rose-derived exosomes.

2. The method for promoting exosome secretion from animal cells according to claim 1, wherein treating animal cells with rose-derived exosomes involves adding the rose-derived exosomes to the culture medium of the animal cells during cultivation and culturing the animal cells.

3. The method for promoting exosome secretion from animal cells according to claim 1 or 2, wherein the rose-derived exosomes are isolated from rose plant cell culture medium, rose callus culture medium, rose plant stem cell culture medium, rose juice, or equivalent rose biological solutions, or are derived from rose plant cells or rose plant stem cells.

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

5. The rose-derived exosomes are isolated from rose plant cell culture medium, rose callus culture medium, rose plant stem cell culture medium, rose juice, or equivalent rose biological solutions, or are derived from rose plant cells or rose plant stem cells, according to claim 4.

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