Cell line for producing FAF1 exosomes at high yield and preparation method thereof

Abstract

According to one aspect of the technology disclosed in this application, the present invention relates to a cell line that stably expresses exosomes loaded with FAF1 protein, and relates to the following: a cell line that can produce exosomes loaded with FAF1 protein in high yield and selectively regulate the expression of FAF1 protein; a method for preparing the cell line; and a method for producing exosomes loaded with FAF1 protein.

Description

[Technical Field]

[0001] The present invention relates to a cell line that can mass-produce FAF1-loaded exosomes while expressing FAF1 only when necessary and stably expressing exosomes, a method for preparing the cell line, and a method for producing FAF1-loaded exosomes.

[0002] Tumor cells are known to be resistant to apoptosis due to genetic mutations related to apoptosis. There are several signaling pathways and receptors involved in apoptosis, but Fas (also known as the Fas receptor, CD95, Apo1, or TNFRSF6) is one of the receptors with superior apoptotic activity. Furthermore, it has been reported that the expression level of Fas-associated factor 1 (FAF1), a protein involved in the Fas pathway, is decreased in various cancers, including lung cancer, colon cancer, liver cancer, prostate cancer, brain cancer, and breast cancer (Xie, Feng, et al. FAF1 phosphorylation by AKT accumulates TGF-B type II receptor and drives breast cancer metastasis. Nature Communications, 2017, 8.1: 1-16).

[0003] Extracellular vesicles (EVVs) are nano-sized vesicles secreted by cells into the external environment for intercellular communication. They are known by various names, such as exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, nanovesicles, and outer membrane vesicles, depending on their origin, secretion mechanism, and size. They contain various biologically active substances, such as proteins, lipids, nucleic acids, and metabolites.

[0004] Among extracellular vesicles, exosomes have attracted attention in recent years in the fields of disease diagnosis and treatment due to their various advantages. Because exosomes are not living cells, they can be stored and transported for long periods of time. Because they do not undergo cell division, the risk of tumorigenesis is low. Furthermore, because exosomes are derived from cells, they have low immunogenicity. Their membrane topology is identical to that of cells, making it easy to deliver drugs or vaccines loaded as cargo to specific cells or tissues. For these reasons, exosomes have recently been utilized in the development of pharmaceuticals for tumor treatment. For example, Korean Patent No. 10-2053065 relates to a pH-sensitive exosome composition using hyaluronic acid and doxorubicin. It discloses a method for preparing pH-sensitive exosomes using doxorubicin and a polymer chemically bound to hyaluronic acid and 3-diethylaminopropylamine, as well as the cancer cell killing effect of exosomes. Furthermore, Korean Patent Publication No. 10-2018-0078173 relates to a novel exosome anticancer agent, and discloses recombinant exosomes that display receptor tyrosine kinase and SIRP (i.e., phagocytosis-promoting protein) on their surface, asparaginase, an anticancer protein, a protein toxin, an antibody or fragment thereof specific to a cancer antigen, a tumor suppressor gene, or an antiangiogenic factor.

[0005] Because the amount of exosomes naturally secreted by cells is very limited, the use of exosomes for tumor treatment requires the development of cell lines capable of mass-producing exosomes at high yields.

[0006] On the other hand, if the protein produced by a cell line induces apoptosis, the protein may have a negative effect on cell survival and proliferation, resulting in a decline in protein productivity as the culture progresses. Therefore, there is a need to develop a cell line and exosome production method that can mass-produce exosomes loaded with apoptosis-inducing proteins without affecting the survival and proliferation of the exosome-producing cells. Summary of the Invention [Problem to be solved by the invention]

[0007] The object of the present invention is to provide a cell line that stably expresses exosomes loaded with FAF1 protein, a method for preparing the cell line, and a method for mass-producing exosomes loaded with FAF1 protein using the cell line. [Means for solving the problem]

[0008] In one aspect, the present invention provides a cell line that stably expresses exosomes loaded with FAF1 protein.

[0009] In another aspect, the present invention provides a method for preparing a cell line that stably expresses exosomes loaded with FAF1 protein, the method comprising: a) introducing a vector containing a gene encoding the FAF1 protein and a promoter regulating the expression of the gene into immune cells, stem cells, somatic cells, plant cells, bacterial cells, yeast cells, mammalian cells, or tumor cells to obtain transformed cells; b) culturing the transformed cells; and c) screening for cells that stably express exosomes loaded with the FAF1 protein among the cultured cells.

[0010] In another aspect, the present invention provides a method for producing exosomes loaded with FAF1 protein, the method comprising the steps of: a) culturing a cell line that stably expresses exosomes loaded with FAF1 protein; and b) isolating the exosomes loaded with FAF1 protein in the cell line culture medium. [Effects of the Invention]

[0011] According to one embodiment of the present invention, a cell line that stably expresses exosomes loaded with FAF1 protein can produce exosomes loaded with FAF1 protein at a high yield, and since it is less affected by apoptosis induced by FAF1 protein, it can mass-produce exosomes loaded with FAF1 protein. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of Western blotting analysis showing the results of inducing FAF1 expression with doxycycline in cells in which pTetOne-FAF1 transduction was confirmed. [Figure 2] FIG. 2 is a graph comparing the viability of the F5-3, F5-4, and F3-1J cell lines with that of the Expi293F cell line. [Figure 3] FIG. 3 is a graph comparing the doubling times of the F5-3, F5-4, and F3-1J cell lines with those of the Expi293F cell line. [Figure 4] FIG. 4 shows changes in the expression of exosome-related markers in the Expi293F cell line and the F5-3 cell line, depending on the presence or absence of doxycycline. [Figure 5] Figure 5 shows the amount of exosomes produced, the amount of FAF1 produced, and the purity of FAF1 in exosomes in the HEK293 cell line, Expi293F cell line, and F5-3 cell line, in the presence or absence of doxycycline. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below. One aspect of the present invention relates to a cell line that stably expresses exosomes loaded with FAF1 protein.

[0014] As used herein, "FAF1" refers to Fas-associated factor 1, which mediates cell necrosis through JNK-dependent mitochondrial dysfunction and contributes to cell proliferation by negatively regulating Aurora-A and inhibiting the G2 / M phase of the cell cycle. FAF1 also binds to ubiquitinated proteins and valosin-containing protein (VCP) and participates in the ubiquitin-proteasome pathway, thereby controlling protein degradation. Unnecessary FAF1 is ubiquitinated via Parkin and degraded via the proteasome pathway.

[0015] FAF1 is involved in various biochemical processes, including cell death, inflammation, cell proliferation, and proteostasis, by activating various pathways. In particular, FAF1 is a tumor suppressor that induces cell death by forming the Fas-death inducing signaling complex (Fas-DISC), plays a role as a tumor suppressor through NF-κB inhibition, and suppresses tumor metastasis through TGF-β signaling.

[0016] As used herein, the term "apoptosis" refers to a type of programmed cell death that can occur in multicellular organisms. Apoptosis is characterized by changes in cell morphology and intracellular biochemical changes that lead to cell death. This process culminates in cell swelling and cracking, alterations to the cell membrane, chromatin condensation and chromosome cleavage, and engulfment by phagocytes. In contrast to necrosis, which is cell death due to acute cell injury, apoptosis does not harm the organism but rather benefits its life cycle. The formation of fingers and toes during differentiation in the human embryo is a prime example of apoptosis. Furthermore, apoptosis is an important mechanism for cell replacement, tissue remodeling, and the removal of damaged cells.

[0017] As used herein, the FAF1 protein may be a polypeptide that comprises the amino acid sequence of Uniprot ID Q9UNN5 or the amino acid sequence of SEQ ID NO:1, and preferably consists of the amino acid sequence of SEQ ID NO:1.

[0018] As used herein, the term "exosome" refers to a vesicle composed of a two-layered phospholipid membrane secreted by cells. Exosomes are known to play an important role in intercellular communication through cell-to-cell signaling, the formation of disease-specific nucleic acids and proteins, and their release into bodily fluids, as well as the intercellular delivery of specific repertoires of nucleic acids, proteins, and lipids important for homeostasis. For example, exosomes are involved in fundamental physiological processes such as neurotransmission, antigen presentation, immune responses, organ development, and fertility, as well as several pathological disorders, including cancer progression, cardiovascular disease, inflammation, and prion transmission.

[0019] Furthermore, exosomes are secreted into the extracellular environment after late endosomes called multivesicular bodies (MVBs) containing intraluminal vesicles (ILVs) fuse with the plasma membrane. MVBs can fuse with the plasma membrane to release ILVs, or they can fuse with lysosomes to degrade their contents. The material present in exosomes differs from the material composition of the cytoplasm, and exosomes can selectively contain RNA and proteins.

[0020] Although exosome production has been reported to be either dependent or independent of the endosomal sorting complex required for transport (ESCRT), the exact mechanism remains unclear. Cells secrete proteins with signal peptides via the endoplasmic reticulum-Golgi complex. Vesicles containing proteins with signal peptides migrate toward the plasma membrane, fuse with it, and export the protein to the extracellular space. However, proteins without signal peptides can be secreted via alternative non-classical secretory pathways. When proteins without signal peptides are secreted, they are secreted either with or without vesicles. Although the exact mechanism of the non-vesicular secretory pathway is unknown, some proteins are secreted via membrane pores or ATP-binding cassette transporters. Vesicular secretion occurs via extracellular vesicles, including exosomes, and occurs via vesicles of various sizes.

[0021] The average diameter of exosomes can be 50 nm to 300 nm, but the present invention is not limited to this.

[0022] Furthermore, in another embodiment of the present invention, the cell line stably expressing exosomes loaded with the FAF1 protein may be a cell line transformed with a vector comprising a gene encoding the FAF1 protein and a promoter regulating the expression of the gene, in which case the FAF1 protein is the same as described above.

[0023] Furthermore, a vector containing a linear puromycin marker gene can be used during transformation to prepare a cell line stably expressing exosomes loaded with the FAF1 protein. The vector containing the linear puromycin marker gene can contain the puromycin marker gene, an SV40 promoter, and an SV40 polyadenylation signal, and is preferably the Tet-One Expression System from Takara Bio Inc. (Japan). TM It can be a linear puromycin marker included in an inducible expression system (Cat. No. 631626).

[0024] As used herein, the term "transformation" refers to a molecular biological technique in which a vector containing an exogenous gene different from that of the original cell is introduced into a cell, and the exogenous gene binds to the DNA present in the original cell, thereby changing the genetic characteristics of the cell.

[0025] The term "vector" as used herein refers to a means for expressing a target gene in a host cell.For example, vectors can be chromosomes, episomes, plasmid vectors, or single-stranded or double-stranded RNA or DNA virus vectors.In addition, vectors include phagemid vectors, cosmid vectors, bacteriophage vectors and virus vectors, such as adenovirus vectors, retrovirus vectors and adeno-associated virus vectors. Vectors that can be used as the above vectors can be prepared by manipulating plasmids commonly used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), phages (e.g., λgt4λB, λCharon, M13, etc.), or viruses (e.g., CMV, SV40, etc.), and are preferably PRSET A. Furthermore, vectors can be introduced into cells using well-known techniques for introducing DNA or RNA into cells.

[0026] Furthermore, in one embodiment of the present invention, the promoter that regulates the expression of a gene encoding a protein may be a promoter that is acted upon by a protein synthesis inhibitor. Protein synthesis inhibitors include aminoglycoside antibiotics, tetracycline antibiotics, macrolide antibiotics, lincosamide antibiotics, streptogramin antibiotics, pleuromutilin antibiotics, phenicol antibiotics, fusidic acid antibiotics, oxazolidinone antibiotics, anisomycin antibiotics, edeine antibiotics, pactamycin antibiotics, puromycin antibiotics, cyclohexamide, aurintricarboxylic acid, diphtheria toxin, ricin, and sodium fluoride. The inhibitor may be selected from the group consisting of, but not limited to, fluoride, sparsomycin, and trichoderma, and any protein synthesis inhibitor having a similar mechanism may be substituted.

[0027] The tetracycline antibiotic may include an antibiotic selected from the group consisting of doxycycline, minocycline, sarecycline, eravacycline, chlortetracycline, demeclocycline, lymecycline, rolitetracycline, omadacycline, methacycline, oxytetracycline, tetracycline, and tigecycline, and may be replaced with any drug known in the art to which the present invention pertains as a tetracycline antibiotic.

[0028] In another embodiment of the present invention, the promoter regulating the expression of the gene encoding the FAF1 protein can be an inducible promoter, such as a nitrogen-deficiency-inducible promoter or a salt-inducible promoter. The inducible promoter can be, for example, a hormone-responsive promoter (e.g., the ecdysone-responsive promoter described in U.S. Pat. No. 6,379,945), a metallothionein promoter (U.S. Pat. No. 6,410,828), or a pathogenesis-related (PR) promoter that can respond to chemicals such as salicylic acid, ethylene, thiamine, and / or BTH (U.S. Pat. No. 5,689,044), or a combination thereof.

[0029] Furthermore, the inducible promoter may be a light-inducible promoter, a metal-inducible promoter, a temperature (low or high temperature)-inducible promoter, a nitrogen-deficiency-inducible promoter, or an antibiotic-inducible promoter, and may be a promoter known in the technical field to which the present invention pertains to induce gene expression depending on the presence or absence of light, metal, temperature change, nitrogen deficiency, antibiotics, etc. Among these, antibiotic-inducible promoters include a thiostrepton-inducible promoter, a tetracycline response element (TRE) promoter, and a Plial promoter, and preferably, the antibiotic-inducible promoter may be a TRE promoter. Furthermore, the TRE promoter is a promoter that is activated by the above-mentioned tetracycline antibiotics.

[0030] In another embodiment of the present invention, a cell line stably expressing exosomes loaded with FAF1 protein may be a cell line in which expression of the gene encoding FAF1 protein is induced in an environment in which a promoter regulating expression of the gene encoding FAF1 protein is activated. Alternatively, a cell line stably expressing exosomes loaded with FAF1 protein may be a cell line in which expression of the gene encoding FAF1 protein is induced in the presence of a protein synthesis inhibitor.

[0031] In one embodiment of the present invention, transformation of cells with a vector is carried out using 1 x 10 cells to be transformed. 5 cells ~1×10 7 This can be done by treating the cells with 1 to 10 μg of pTetOne-FAF1 plasmid (Takara Bio Inc., Japan). Preferably, 1 × 10 cells are transformed. 6 The cells can be cultured by treating them with 5 μg of pTetOne-FAF1 plasmid, but the present invention is not limited thereto.

[0032] In another embodiment of the present invention, the cell line expressing exosomes loaded with FAF1 protein can be derived from immune cells, stem cells, somatic cells, plant cell lines, bacterial cell lines, yeast cell lines, mammalian cell lines or tumor cells.

[0033] The immune cell may be selected from the group consisting of, but is not limited to, a dendritic cell, a natural killer cell, a T cell, a B cell, a regulatory T cell (Treg cell), a natural killer T cell, an innate lymphoid cell, a macrophage, a granulocyte, a chimeric antigen receptor-T cell (CAR-T), a lymphokine-activated killer cell (LAK), and a cytokine-induced killer cell (CIK).

[0034] Stem cells may be, but are not limited to, mesoderm stem cells, pluripotent stem cells, multipotent stem cells, or unipotent stem cells. Pluripotent stem cells may be, but are not limited to, embryonic stem cells (ES cells), embryonic germ cells (EG cells), or induced pluripotent stem cells (iPS cells, iPSCs). Multipotent stem cells include, but are not limited to, mesenchymal stem cells (e.g., adipose-derived, bone marrow-derived, umbilical cord blood-derived, or umbilical cord-derived), hematopoietic stem cells (e.g., bone marrow-derived or peripheral blood-derived), neural stem cells, and adult stem cells such as germ stem cells. Mesenchymal stem cells (MSCs) can be, but are not limited to, human embryonic stem cell-derived mesenchymal stem cells (hES-MSCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), umbilical cord-derived mesenchymal stem cells (UC-MSCs), or adipose-derived mesenchymal stem cell-conditioned medium (ADSCs).

[0035] Furthermore, stem cells may be autologous or allogeneic, may be derived from any animal, including humans and non-human mammals, and may be, but are not limited to, embryonic stem cells, adult stem cells, or induced pluripotent stem cells. The term "embryonic stem cells" refers to cells extracted during embryonic development and can be obtained by removing the inner cell mass from a blastocyst embryo just before the fertilized egg implants in the mother's uterus and culturing it in vitro. Embryonic stem cells refer to pluripotent or totipotent cells with the ability to self-renew and differentiate into cells of all tissues of an individual, and in a broad sense include embryoid bodies derived from embryonic stem cells.

[0036] The somatic cells may be selected from the group consisting of fibroblasts, chondrocytes, synoviocytes, keratinocytes, adipocytes, osteoblasts, osteoclasts and peripheral blood mononuclear cells.

[0037] Tumor cells can be derived from, but are not limited to, human ovarian cancer cell lines (SKOV3 and OVCAR3), human breast cancer cell lines (MCF-7, T47D, BT-474, and MDA-MB-231), human liver carcinoma cell lines (Hep3B and HepG2), human glioblastoma cell lines (U87MG and U251), human colorectal cancer cell lines (SW480, HT-29, HCT116, and Caco-2), human lung cancer cell lines (A549, NCIH358, and NCI-H460), human prostate cancer cell line (22RV1), human cervical cancer cell line (HeLa), human melanoma cell line (A375), and human gastric cancer cell line (NCI-N87).

[0038] As the plant cell line, bacterial cell line, or yeast cell line, any cell line known in the art to which the present invention belongs to as being suitable for protein production can be used.

[0039] In another embodiment of the present invention, the cell line expressing exosomes loaded with FAF1 protein may be derived from a mammalian cell line, such as a CHO cell line, an NS0 cell line, an Sp2 / 0 cell line, a BHK cell line, a C127 cell line, a HEK293 cell line, a HEK293T cell line, a HEK-293 STF cell line, a 293T / 17 cell line, a 293T / 17 SF cell line, a HEK-293.2sus cell line, a HEK-293 F cell line, an HT-1080 cell line, a PER.C6 cell line, a NuLi-1 cell line, an ARPE-19 cell line, a VK2 / E6E7 cell line, an Ect1 / E6E7 cell line, a RWPE-2 cell line, a WPE-stem cell line, an End1 / E6E7 cell line, a WPMY-1 cell line, an NL20 cell line, an NL20-TA cell line, or a WT The cell line expressing exosomes carrying FAF1 protein may be selected from the group consisting of, but not limited to, 9-7 cell line, WPE1-NB26 cell line, WPE-int cell line, RWPE2-W99 cell line, Expi293F cell line, and BEAS-2B cell line. Preferably, the cell line expressing exosomes carrying FAF1 protein may be derived from Expi293F cells, which are suspension cells.

[0040] In another embodiment of the present invention, a cell line stably expressing exosomes loaded with FAF1 protein can increase the number of exosomes, the amount of FAF1 protein loaded on exosomes, and the proportion of FAF1 among proteins loaded on exosomes.

[0041] In one embodiment of the present invention, a cell line that stably expresses exosomes loaded with FAF1 protein can be a cell line in which the amount of exosomes loaded with FAF1 protein obtained by cell culture in an environment in which a promoter that regulates the expression of a gene encoding the FAF1 protein is activated is increased compared to the amount of exosomes loaded with FAF1 protein obtained by cell culture in an environment in which the promoter is inactivated.

[0042] Furthermore, a cell line stably expressing exosomes loaded with FAF1 protein may be a cell line in which the amount of FAF1 protein-loaded exosomes obtained by cell culture in an environment in which a promoter regulating the expression of the gene encoding FAF1 protein is activated is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, or at least 15-fold compared to the amount of FAF1 protein-loaded exosomes obtained by cell culture in an environment in which the promoter is not activated. Preferably, a cell line stably expressing exosomes loaded with FAF1 protein may be a cell line in which the amount of FAF1 protein-loaded exosomes obtained by cell culture in an environment in which a promoter regulating the expression of the gene encoding FAF1 protein is activated is increased by at least 3-fold, at least 5-fold, at least 6-fold, or at least 12-fold compared to the amount of FAF1 protein-loaded exosomes obtained by cell culture in an environment in which the promoter is not activated.

[0043] In another embodiment of the present invention, a cell line stably expressing exosomes loaded with FAF1 protein may be a cell line in which the content of exosomes loaded with FAF1 protein obtained by cell culture in the presence of a protein synthesis inhibitor is increased compared to the content of exosomes loaded with FAF1 protein obtained by cell culture in the absence of a protein synthesis inhibitor.

[0044] Furthermore, a cell line that stably expresses exosomes loaded with FAF1 protein can be a cell line in which the amount of exosomes loaded with FAF1 protein obtained by cell culture in the presence of a protein synthesis inhibitor is increased by 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 11-fold or more, 12-fold or more, 13-fold or more, 14-fold or more, or 15-fold or more compared to the amount of exosomes loaded with FAF1 protein obtained by cell culture in the absence of a protein synthesis inhibitor.

[0045] In another embodiment of the present invention, a cell line stably expressing exosomes loaded with FAF1 protein may be a cell line in which the proportion of FAF1 among exosomal proteins is increased compared to an HEK293 cell line transformed with a vector containing a gene encoding the FAF1 protein.Furthermore, a cell line stably expressing exosomes loaded with FAF1 protein may be a cell line in which the proportion of FAF1 among exosomal proteins is increased by at least two-fold, at least three-fold, at least four-fold, at least five-fold, and preferably at least three-fold compared to an HEK293 cell line transformed with a vector containing a gene encoding the FAF1 protein.

[0046] In yet another embodiment of the present invention, a cell line stably expressing exosomes loaded with FAF1 protein may be a cell line in which the number of exosomes loaded with FAF1 protein, the proportion of FAF1 among proteins in exosomes, or both, is increased compared to a HEK293 cell line transformed with a vector containing a gene encoding the FAF1 protein.

[0047] In one aspect, the present invention relates to exosomes obtained from a cell line that stably expresses exosomes loaded with FAF1 protein.

[0048] Exosomes can be obtained by methods commonly used in the art for isolating exosomes, such as size exclusion chromatography, ion exchange chromatography, density gradient centrifugation, differential centrifugation, ultrafiltration, tangential flow filtration, exosome precipitation, total exosome extraction kits, immunoabsorbent capture, affinity methods such as affinity capture or affinity purification, immunoassays, microfluidic separation, or a combination thereof.

[0049] According to a specific embodiment of the present invention, the culture medium of a cell line stably expressing exosomes loaded with FAF1 protein is collected, centrifuged at 300 g for 10 minutes, 2,000 g for 10 minutes, and 10,000 g for 30 minutes to separate the supernatant, filtered using a 0.2 μm filter, and centrifuged at 150,000 g for 70 minutes using an ultracentrifuge. Alternatively, the supernatant obtained by centrifugation may be removed, the pellet washed by adding PBS, and then centrifuged again at 150,000 g for 70 minutes, the supernatant removed, and the exosomes remaining in the lower layer isolated; however, the present disclosure is not limited thereto.

[0050] In the present invention, exosomes obtained from a cell line that stably expresses exosomes loaded with FAF1 protein can be stored at -20°C to -80°C.

[0051] Furthermore, in the present invention, the FAF1 protein can be secreted into the extracellular space via exosomes, and the extracellularly secreted FAF1 protein can induce apoptosis in other cells.

[0052] In one embodiment, the present invention provides a method for preparing a cell line stably expressing exosomes loaded with FAF1 protein, the method comprising the steps of: (a) transducing a vector containing a gene encoding FAF1 protein and a promoter regulating gene expression into cells selected from the group consisting of immune cells, stem cells, somatic cells, plant cells, bacterial cells, yeast cells, mammalian cells, and tumor cells to obtain transformed cells; (b) culturing the transformed cells; and (c) screening the cultured cells for cells stably expressing exosomes loaded with FAF1 protein. The immune cells, stem cells, somatic cells, tumor cells, FAF1 protein, promoter regulating expression of the gene encoding FAF1 protein, vector, and transformation are the same as those described above. Furthermore, the terms "plant cells," "bacterial cells," "yeast cells," and "mammalian cells" refer to cells belonging to or derived from the above-mentioned plant cell lines, bacterial cell lines, yeast cell lines, and mammalian cell lines, respectively.

[0053] The term "culture" as used herein refers to a method for growing cells or microorganisms under appropriate artificially controlled environmental conditions. In the present invention, the method for culturing transformed cells can be carried out using methods widely known in the art.

[0054] Furthermore, media that can be used to culture cells in the present invention are known media used for culturing cells. Examples of media include commercially produced media such as Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, Dulbecco's Modified Eagle's Medium:Nutrient Mixture F-10 (DMEM / F-10), Dulbecco's Modified Eagle's Medium:Nutrient Mixture F-12 (DMEM / F-12), α-Minimal essential Medium (α-MEM), Glasgow's Minimal Essential Medium (G-MEM), Iscove's Modified Dulbecco's Medium (IMDM), KnockOut DMEM, and Essential Medium. The medium may include, but is not limited to, Essential 8 Medium (E8), Expi293F expression medium, synthetic medium, or a medium selected from the group consisting of commercially available serum-free medium, protein-free medium, and chemically defined medium. Furthermore, the medium may contain the above-mentioned protein synthesis inhibitors.

[0055] In another embodiment of the present invention, a method for producing exosomes loaded with FAF1 protein can include culturing a cell line stably expressing exosomes loaded with FAF1 protein and isolating the exosomes loaded with FAF1 protein from the cell line culture medium. The above isolation method can be used to isolate exosomes, and preferably, the isolation of exosomes can be performed using a centrifuge.

[0056] The expression level of a protein can be used as a marker for selecting a cell line to be used in the present invention, and protein expression can be measured using methods known in the art. For example, antibodies that specifically detect proteins may be commercially available and can be used to detect proteins using Western blot, co-immunoprecipitation, immunofluorescence, enzyme-linked immunosorbent assay, etc. The expression level of a particular protein can be specifically identified using such antibodies, but the present invention is not limited thereto. [Example]

[0057] The present invention will be described in detail below with reference to examples. However, the following examples are for the purpose of illustrating the present invention, and the present invention is not limited thereto.

[0058] Example 1: Creation of a cell line capable of producing FAF1 exosomes at a high yield

[0059] Example 1.1. Creation of a cell line stably expressing FAF1 exosomes derived from the Expi293F cell line

[0060] [Cell culture] The Expi293F cell line (Gibco, USA) was purchased as the base cell for generating a cell line stably producing exosomes loaded with the FAF1 protein (hereafter referred to as the FAF1 exosome stable-producing cell line). The Expi293F cell line (hereafter referred to as Expi293F cells) is a suspension cell derived from the human embryonic kidney (HEK) 293 cell line. It is known to be capable of high-density culture, highly transducible, and to exhibit superior protein yields compared to HEK 293 cells (Thermo Fisher Scientific Expi293F cell line product description, https: / / www.thermofisher.com / order / catalog / product / A14527). Furthermore, Expi293F cells can be cultured in Expi293 Expression Medium (Gibco, USA), a serum- and external protein-free synthetic medium, to eliminate exosomes or external proteins contained in serum during exosome production (Thermo Fisher Scientific Expi293 Expression Medium product description, https: / / www.thermofisher.com / order / catalog / product / A1435101).

[0061] Expi293F cells were cultured in cell culture Erlenmeyer flasks at 3 × 10 in Expi293 expression medium (Gibco, USA). 5 cells / mL or 4 x 10 5 Dispense at a concentration of 3 x 10 cells / mL. 6 Suspension cells were cultured until the number of cells / mL reached 100. Expi293F cells were maintained by subculture in Expi293 expression medium once every 3-4 days under conditions of 8% CO2, 37°C, and 125 rpm.

[0062] For transduction, suspension cells were adapted to adherent cells and used. 1 × 10 6Expi293F cells were plated onto 100 mm culture dishes and cultured in Dulbecco's Modified Eagle's Medium (DMEM, WelGENE, Korea) containing 10% fetal bovine serum (FBS, Atlas Biologicals, USA) and antibiotic-antimycotic (penicillin / streptomycin, Gibco, USA) at 37°C in 5% CO2. After at least 24 hours, the cells adhered to the culture dish and their morphology changed (adaptation of suspension cells to adherent cells).

[0063] Meanwhile, the adapted Expi293F cells were maintained in DMEM medium containing antibiotics, 10% FBS (Atlas Biologicals, USA), and 1% antibiotic-antimycotic (penicillin / streptomycin, Gibco, USA) at 5% CO2 and 37°C by subculturing once every 3–4 days.

[0064] 1 x 10 cells adapted to adherent cells 6 3 x 10 Expi293F cells, adapted to adherent cells, were dispensed into a 100 mm culture dish. 5 Expi293F cells were plated onto a 60-mm culture dish and then cultured in Dulbecco's Modified Eagle's Medium (DMEM, WelGENE, Korea) containing 10% FBS at 37°C and 5% CO for 24 hours.

[0065] [Transduction] The pTetOne-FAF1 plasmid was constructed using the pTetOne plasmid (TaKaRa Bio, Japan) and the FAF1 amino acid sequence of SEQ ID NO: 1. The pTetOne plasmid (TaKaRa Bio, Japan) lacking FAF1 was used as a control. The pTetOne-FAF1 plasmid / control plasmid (3 μg or 5 μg) and a puromycin linear marker (100 ng, Takara Bio, Japan) were mixed with Opti-MEM medium (Thermo Fisher Scientific, USA) to prepare a plasmid-puromycin linear marker-medium mixture with a total volume of 200 μL. The resulting mixture was incubated at room temperature for 5 minutes. ExpiFectamine (16 μL, Thermo Fisher Scientific, USA) was mixed with Opti-MEM medium to prepare a mixture with a total volume of 240 μL. The resulting mixture was incubated at room temperature for 5 minutes. The plasmid-puromycin linear marker-medium mixture and the ExpiFectamine-medium mixture were then mixed and incubated at room temperature for 10 minutes.

[0066] The completed reaction mixture was added to Expi293F cells that had been cultured in DMEM medium in a culture dish for 24 hours, and the reaction was continued for 24 hours under conditions of 5% CO2 and 37°C to transduce the cells with the pTetOne-FAF1 plasmid.

[0067] Example 1.2. Screening for cell lines stably producing FAF1 exosomes

[0068] Cells transfected with the pTetOne-FAF1 plasmid prepared in Example 1.1 were cultured in the same culture dish with DMEM medium containing 10% FBS and puromycin, which was replaced every 2–3 days. Puromycin was added to the medium at a concentration of 1 μg / mL or 3 μg / mL, taking into account the rate of apoptosis.

[0069] One to two weeks after transduction with the pTetOne-FAF1 plasmid, untransduced cells lacking puromycin resistance were killed. When puromycin-resistant transduced cells formed colonies, the colonies were isolated using cloning cylinders (Sigma-Aldrich, USA) and cultured in 35 mm culture dishes to obtain 13 puromycin-resistant cell lines (F3-2, F3-3, F3-4, F5-1, F5-2, F5-3, F5-4, F5-5, F5-6, F5-7, F3-1J, F5-2J, and F5-3J).

[0070] Next, to confirm whether the 13 puromycin-resistant cell lines expressed FAF1 after transduction with the TetOne system, doxycycline was added and the FAF1 expression levels were measured by Western blot.

[0071] Specifically, F3-2, F3-3, F3-4, F5-1, F5-2, F5-3, F5-4, F5-5, F5-6, F5-7, F3-1J, F5-2J, and F5-3J cell lines were each cultured at 3 × 10 5 The cells were then plated in duplicate onto 60 mm culture dishes and cultured for 3 days in 10% FBS DMEM medium with or without 100 ng / mL doxycycline. Given the half-life of doxycycline in the medium (1 day), the medium was changed once daily. After 3 days, the cultured cells were harvested and the expression levels of FAF1 in doxycycline-treated cells were compared with those in untreated cells by Western blot analysis.

[0072] To perform Western blotting, F3-2, F3-3, F3-4, F5-1, F5-2, F5-3, F5-4, F5-5, F5-6, F5-7, F3-1J, F5-2J, and F5-3J cell cultures were centrifuged at 300 g for 10 minutes, and the supernatant was removed to obtain cell pellets. Mammalian lysis buffer (80 μL) was added to the cell pellets, and the cells were lysed on ice for 30 minutes. Protein in the cell pellets was diluted with Bradford solution, and its concentration was measured at 595 nm using a spectrophotometer (MECASYS, Korea). An aliquot of the lysed pellet was mixed with 5 μL of SDS sample buffer to obtain 20 μg of protein, and the mixture was incubated in boiling water for 3 minutes. A 10% acrylamide gel was prepared using Western blot (Amersham Biosciences, UK). A 3-minute boiled cell protein sample was placed on the gel, and proteins were separated by size at 80–100 V. The acrylamide gel containing the separated proteins was then placed on a nitrocellulose membrane (GE Healthcare, USA). Proteins were transferred to the nitrocellulose membrane at 200 mA for 2 hours. The membrane was then blocked with skim milk and coated with an anti-FAF1 antibody (E-4, Santacruz, 1:1000, mouse IgG) followed by a secondary antibody (HRP-anti-mouse, 31439, Thermo Fisher Scientific, 1:1000, goat IgG). FAF1 protein levels were confirmed using a Western blot detection kit (AbFrontier, Korea) and a ChemiDoc-It Imaging System (UVP, USA). Western blot results were quantified using ImageJ. β-actin and GAPDH were used as loading controls.

[0073] The selection criteria for stable FAF1 exosome-producing cell lines were cells that produced more than three times as much FAF1 as cells not treated with doxycycline. Three types of cells (F5-3, F5-4, and F3-1J) met the selection criteria for stable FAF1 exosome-producing cell lines (Figure 1). Compared to cells not treated with doxycycline, F5-3 produced 13.2 times more FAF1, F5-4 produced 6.7 times more FAF1, and F3-1J produced 5.1 times more FAF1. For the F5-3, F5-4, and F3-1J cell lines, 1 × 10 7 The cells were maintained in Expi293 expression medium (Gibco, USA) in cell culture Erlenmeyer flasks under conditions of 8% CO2, 37°C, and 125 rpm, with subculture every 3–4 days, allowing them to adapt to suspension cell life. This established a stable cell line producing FAF1 exosomes with on / off control.

[0074] Example 1.3. Cell viability and doubling time of a cell line stably expressing on / off-controllable FAF1 exosomes

[0075] The FAF1 exosome-stably expressing cell lines F5-3, F5-4, and F3-1J, which can be turned on / off, were subcultured under the conditions of Example 1.2. Among these FAF1 exosome-stably expressing cell lines, those with slow cell division rates were dispensed into Erlenmeyer flasks at a cell concentration of 6 × 10 5 cells / mL or 8 x 10 5 Set the platelet count to 100 cells / mL and ensure that the cell concentration reaches 3 x 10 cells / mL within 3-4 days. 6 The culture was carried out under the conditions of Example 1.2 to reach a concentration of 1000 cells / mL.

[0076] The Expi293F cell line was maintained in Expi293 expression medium (Gibco, USA) under conditions of 8% CO 2 , 37°C and 125 rpm by subculturing once every 3 to 4 days.

[0077] The subcultured cell culture medium (10 μL) was separated and mixed with 140 μL of Muse Count & Viability Kit (Luminex, USA), incubated at room temperature for 5 minutes, and then placed in a Muse cell analyzer (Luminex, USA) to measure cell count and viability.

[0078] Using the cell numbers measured during subculture, the doubling time was calculated using the following formula:

[0079]

number

[0080] Expi293F, F5-3, F5-4, and F3-1J cells were cultured for more than 10 passages and their viability and cell concentration were measured using the Muse cell analyzer (Figures 2 and 3). The average viability of each cell line was measured as follows: 95.4 ± 1.12% for Expi293F cells, 90.8 ± 2.63% for F5-3 cells, 92.0 ± 2.64% for F5-4 cells, and 90.7 ± 4.53% for F3-1J cells. Although the viability of the FAF1 exosome-stably expressing cell line was slightly lower than that of the Expi293F cell line, all cell lines showed good viability of over 90%.

[0081] The doubling time of each cell line was calculated using the measured cell concentrations: 24.4 ± 1.75 h for Expi293F cells, 33.8 ± 4.76 h for F5-3 cells, 27.7 ± 3.40 h for F5-4 cells, and 30.4 ± 4.17 h for F3-1J cells. The doubling time of the FAF1 exosome-stably expressing cell lines was slightly increased compared to the Expi293F cell line. The lower viability and longer doubling time compared to the original Expi293F cell line are likely due to the inability of the transduced TetOne-FAF1 system to completely suppress the expression of apoptosis-inducing FAF1.

[0082] <Example 2> Characterization of exosomes derived from FAF1 exosome stably expressing cell lines

[0083] Example 2.1. Isolation of exosomes using an ultracentrifuge

[0084] Exosomes from cell lines stably expressing FAF1 and the Expi293F cell line were isolated using an ultracentrifuge (Optima XE-100, BECKMAN Coulter, USA).

[0085] Specifically, 3 x 10 6 Expi293F, F5-3, F5-4, and F3-1J cells were cultured at a concentration of 1 / mL in 50 mL of Expi293 expression medium containing 1 μg / mL doxycycline for 48 or 72 hours, respectively. To isolate all exosomes within the culture period, the medium was not changed during the culture period. The medium for each cell was collected and centrifuged at 300 g for 10 minutes, 2,000 g for 10 minutes, and 10,000 g for 30 minutes to separate the supernatant, which was then filtered through a 0.2 μm syringe filter (BioFACT, Korea).

[0086] To isolate exosomes for use in experiments, the supernatant was centrifuged at 150,000 g for 70 minutes in a BECKMAN Coulter optima XE-100 ultracentrifuge using a 45Ti rotor. The supernatant was discarded, and the pellet was washed with PBS. The washed PBS was centrifuged again at 150,000 g for 70 minutes. The supernatant was discarded, and the remainder was collected in an EP tube using PBS.

[0087] Isolated exosomes should be stored at -80°C and used within one week of isolation, or thawed and stored at 4°C.

[0088] Example 2.2. Confirmation of FAF1 exosome stably expressing cells and the loading of FAF1 and exosome-associated markers in isolated exosomes

[0089] To examine whether the exosomes isolated from the FAF1 exosome-stably expressing cell line in Example 1.2 above are identical to the exosomes isolated from the parent cell line, the Expi293F cell line, the amount of protein isolated from Expi293F cells and exosomes isolated therefrom was compared by Western blotting with the amount of protein isolated from F5-3 cells, which showed the highest FAF1 expression level in the Western blotting experiment in Example 1.3, and exosomes isolated therefrom.

[0090] Specifically, cells and exosomes were isolated from a control group in which Expi293F cells and F5-3 cells were cultured for 48 hours without doxycycline, and from experimental groups in which cells were treated with 1 μg / mL of doxycycline and cultured for 48 or 72 hours, using the method described in Example 2.1.

[0091] To perform Western blotting, the cell culture medium was centrifuged at 300 g for 10 minutes, the supernatant was removed, and the cell pellet was obtained. Mammalian lysis buffer (50 μL) was added to the cell pellet and lysed on ice for 30 minutes.

[0092] The dissolved pellet was mixed with 5 μL of SDS sample buffer and incubated in boiling water for 3 minutes. For the exosomes isolated in Example 2.1, 5 μL of SDS sample buffer was mixed with the exosomes and incubated in boiling water for 3 minutes. An 8-10% acrylamide gel was prepared using a Western blot kit (Amersham Biosciences, UK). To perform Western blots of equal amounts of protein, the intracellular proteins were diluted with Bradford solution, and then the concentration was measured at 595 nm using a spectrophotometer (MECASYS, Korea).

[0093] The concentration of exosomes was measured using nanoparticle tracking analysis (NTA), and the isolated exosomes were measured at a concentration of 1 × 10 8 Particles / mL ~ 1 x 10 9 The exosomes were diluted to a concentration of 5 × 10 particles / mL and the exosome concentration was measured using an NS300 (Malvern Panalytical, UK). 9 Exosomes were placed on an acrylamide gel. After boiling for 3 minutes, a sample of cells and exosome proteins was placed on the gel. Proteins were separated by size using a voltage of 80–100 V. The acrylamide gel containing the separated proteins was placed on a nitrocellulose membrane, and the proteins were transferred to the membrane using a current of 200 mA for 2 hours. The nitrocellulose membrane was blocked with skim milk and then coated with the primary antibody listed in Table 1 below, followed by the secondary antibody listed in Table 1 below. The amounts of FAF1 and exosome-associated marker proteins (Alix, CD81, CD47, CD9, and syntenin-1) were confirmed using a Western blot detection kit (AbFrontier, Korea) and a ChemiDoc-It imaging system (UVP, USA). GAPDH was used as a loading control.

[0094] [Table 1]

[0095] Depending on the antibody, cellular protein (10 or 20 μg) was added and Western blot was performed. To quantify the amount of FAF1 loaded onto exosomes by Western blot, a predetermined amount of recombinant human FAF1 protein (BOSTER, USA) was combined and subjected to Western blot analysis, analyzed using ImageJ, and the measured values ​​were compared with those of FAF1 in exosomes.

[0096] Intracellular FAF1 was significantly increased only when F5-3 cells were treated with doxycycline, and the amount of FAF1 loaded onto exosomes was also significantly increased when F5-3 cells were treated with doxycycline (Figure 4, F5-3 cells and F5-3 Exo). In particular, the highest amount of FAF1 was loaded when the cells were cultured for 72 hours. All exosome-associated markers were present in exosomes, confirming that the protein is an exosomal protein.

[0097] Example 2.3. Comparison of exosome number and FAF1 loading between HEK293 cell line and FAF1 exosome stably expressing cell line

[0098] We compared the differences in FAF1-loaded exosome production among HEK293 cells transduced with 3xFlag tagged-FAF1 plasmid / control plasmid and cultured for 24 hours, Expi293F cells cultured for 48 hours without doxycycline, Expi293F cells treated with 1 μg / mL doxycycline and cultured for 48 hours, F5-3 cells cultured for 48 hours without doxycycline, and F5-3 cells treated with 1 μg / mL doxycycline and cultured for 48 hours.

[0099] HEK293 cells were maintained in DMEM medium containing 10% FBS and antibiotics under conditions of 5% CO 2 and 37° C. by subculturing once every 3 to 4 days.

[0100] To transiently overexpress FAF1 in HEK293 cells, 3xFlag tagged-FAF1 plasmid / control plasmid (8 μg) was added to 6.5x10 6 HEK293 cells were transduced with the 3xFlag tagged FAF1 vector. Specifically, HEK293 cells were plated onto 150 mm culture dishes and cultured in DMEM medium containing 10% FBS at 37°C in 5% CO2 for 24 hours. 3xFlag tagged FAF1 vector was prepared according to a previously described method (Yu et al., FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration. Cell Death & Differentiation, 2016, 23.11: 1873-1885). DNA and BioT (Bioland Scientific, USA) were mixed at a ratio of 1:1.5 with DMEM medium without FBS or antibiotics to prepare a total volume of 180 μL, and the mixture was incubated at room temperature for 5 minutes. The mixture was then added to the cells cultured for 24 hours and incubated at 37°C in 5% CO2 for 24 hours.

[0101] To isolate exosomes, the medium of transduced HEK293 cells was replaced with DMEM medium lacking FBS and antibiotics, and the cells were cultured for 24 hours. Because long-term culture of HEK293 cells transiently overexpressing FAF1 without FBS led to apoptosis, the cells were cultured for only 24 hours, and exosomes were isolated using the method described in Example 2.1.

[0102] Expi293F and F5-3 cells were cultured in Expi293 expression medium or Expi293 expression medium containing 1 μg / mL doxycycline.

[0103] The amount of FAF1 loaded onto exosomes isolated from HEK293 cells, Expi293F cells, and F5-3 cells was quantified by Western blot and ELISA.

[0104] When quantifying exosomes by Western blotting in Example 2.2, recombinant FAF1 protein, which accurately measures the amount of protein, was also loaded, and the protein bands were measured using ImageJ and quantified by comparison.

[0105] ELISA analysis was performed using a human FAF1 ELISA kit (Abbexa, UK). Exosomes isolated from HEK293, Expi293F, and F5-3 cells were lysed in mammalian lysis buffer (10 μL) on ice for 30 minutes. Standard / sample dilutions were then added to each well of the 96-well plate. For HEK293 exosomes, 1 × 10 exosomes were added per well. 8 pcs or 1 x 10 7 1 × 10 for Expi293F and F5-3 exosomes 9 pcs or 1 x 10 8 The exosomes were diluted to a concentration of 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.13 ng / mL, 1.56 ng / mL, and 0.78 ng / mL. The FAF1 standard and diluted exosomes were combined and dispensed in 100 μL aliquots into a 96-well plate of the ELISA kit and incubated at 37°C for 2 hours. The supernatant was discarded, and solution A from the ELISA kit was diluted with the entire solution. 100 μL aliquots were dispensed and incubated at 37°C for 1 hour. After discarding the supernatant, the plates were washed twice with washing buffer. After discarding the supernatant, solution B from the ELISA kit was diluted with the entire solution. 100 μL aliquots were dispensed and incubated at 37°C for 1 hour. After discarding the supernatant, the plates were washed three times with washing buffer. After discarding the supernatant, 90 μL of TMB solution was dispensed into each well and incubated at 37°C for 20–30 minutes, followed by 50 μL of stop solution. Each well showed a color change corresponding to the amount of protein present, which was then measured at 450 nm using a microplate reader (PerkinElmer, USA) to quantify FAF1 protein. The FAF1 loading amount and exosome number measured by Western blot and ELISA were combined to derive average values.

[0106] Furthermore, the proportion of FAF1 in the total protein per exosome was measured. Exosomal proteins were dissolved in mammalian lysis buffer, exposed to the exosomes, diluted with Bradford solution, and the amount of protein was measured at a wavelength of 595 nm using a spectrophotometer (MECASYS, Korea).

[0107] Figure 5 shows FAF1 loading, measured exosome numbers, and the percentage of FAF1 in total protein per exosome.

[0108] The number of exosomes produced per 50 mL of culture medium was at least 3.7-fold higher in Expi293F and F5-3 cells than in HEK293 cells. This means that when using the same medium, Expi293F and F5-3 cells can be cultured at higher densities, thereby enabling the production of more exosomes at a time. Comparing the yield of FAF1-loaded exosomes, doxycycline-induced F5-3 cells produced 71.2-fold more exosomes than HEK293 cells.

[0109] Based on 50 mL of culture medium, the total amount of FAF1 protein loaded onto exosomes was measured to be 70 ng for HEK293 exosomes and 2,287 ng for F5-3 exosomes, confirming that F5-3 cells produced 32.7 times more FAF1 protein than HEK293 cells. Although the amount of FAF1 loaded per exosome was less in F5-3 cells than in HEK293 cells, when the same volume of culture medium was used, the total amount of FAF1 that could be produced at one time was 32.7 times greater in F5-3 cells than in HEK293 cells, indicating that more FAF1 can be delivered to the target.

[0110] Comparing the amount of FAF1 protein measured in each cell line with the amount of exosomal protein, the amount was 0.41% in HEK293 cells transduced with 3xFlag tagged-FAF1, while it was 1.43% in F5-3 cells cultured for 48 hours after treatment with 1 μg / mL doxycycline. This indicates that exosomes isolated from HEK293 cells contain more impurities than F5-3 cells due to the presence of not only FAF1 protein but also various other proteins.

[0111] Therefore, it was confirmed that exosomes isolated from F5-3 cells produce more FAF1 protein and produce more pure exosomes when the same volume of medium is used.

Claims

1. A cell line that stably expresses exosomes loaded with the FAF1 protein.

2. The cell line of claim 1, wherein the cell line is transformed with a vector comprising a gene encoding the FAF1 protein and a promoter that regulates the expression of the gene.

3. The cell line of claim 2, wherein expression of the gene encoding the FAF1 protein is induced in an environment in which a promoter regulating expression of the gene is activated.

4. The cell line of claim 2, wherein the FAF1 protein comprises the amino acid sequence of SEQ ID NO:

1.

5. The cell line of claim 2, wherein the promoter regulating the expression of the gene encoding the FAF1 protein is activated by a protein synthesis inhibitor.

6. Protein synthesis inhibitors include aminoglycoside antibiotics, tetracycline antibiotics, macrolide antibiotics, lincosamide antibiotics, streptogramin antibiotics, pleuromutilin antibiotics, phenicol antibiotics, fusidic acid antibiotics, oxazolidinone antibiotics, anisomycin antibiotics, edeine antibiotics, pactamycin antibiotics, puromycin antibiotics, cyclohexamide, aurintricarboxylic acid, diphtheria toxin, ricin, and sodium fluoride.

6. The cell line of claim 5, wherein the cell line is selected from the group consisting of: Bacillus subtilis, Bacillus fluoride, Bacillus sparsomycin, and Bacillus trichoderma.

7. 7. The cell line of claim 6, wherein the tetracycline antibiotic is selected from the group consisting of doxycycline, minocycline, sarecycline, eravacycline, chlortetracycline, demeclocycline, lymecycline, rolitetracycline, omadacycline, methacycline, oxytetracycline, tetracycline, and tigecycline.

8. The cell line according to claim 5, wherein the promoter that regulates the expression of the gene encoding the FAF1 protein is a tetracycline response element (TRE) promoter.

9. The cell line of claim 2, wherein a vector containing a linear puromycin marker gene is further used during transformation.

10. 2. The cell line of claim 1, which is derived from an immune cell, a stem cell, a somatic cell, a mammalian cell line, or a tumor cell.

11. Mammalian cell lines include CHO cell line, NS0 cell line, Sp2 / 0 cell line, BHK cell line, C127 cell line, HEK293 cell line, HEK293T cell line, HEK-293 STF cell line, 293T / 17 cell line, 293T / 17 SF cell line, HEK-293.2sus cell line, and HEK-293 11. The cell line of claim 10, which is a cell line selected from the group consisting of: HT-1080 cell line, PER.C6 cell line, NuLi-1 cell line, ARPE-19 cell line, VK2 / E6E7 cell line, Ect1 / E6E7 cell line, RWPE-2 cell line, WPE-stem cell line, End1 / E6E7 cell line, WPMY-1 cell line, NL20 cell line, NL20-TA cell line, WT9-7 cell line, WPE1-NB26 cell line, WPE-int cell line, RWPE2-W99 cell line, Expi293F cell line, and BEAS-2B cell line.

12. The cell line of claim 10, wherein the mammalian cell line is the Expi293F cell line.

13. The cell line according to claim 2, wherein the amount of exosomes carrying FAF1 protein obtained by cell culture in an environment in which a promoter that regulates the expression of a gene encoding the FAF1 protein is activated is 2 to 15 times greater than the amount of exosomes carrying FAF1 protein obtained by cell culture in an environment in which the promoter is not activated.

14. The cell line of claim 1, wherein the proportion of FAF1 among proteins in exosomes is increased by at least three times compared to a HEK293 cell line transformed with a vector containing a gene encoding the FAF1 protein.

15. The cell line described in claim 1, in which the number of exosomes carrying FAF1 protein, the proportion of FAF1 among proteins in exosomes, or both, is increased compared to a HEK293 cell line transformed with a vector containing a gene encoding FAF1 protein.

16. A method for preparing a cell line that stably expresses exosomes loaded with FAF1 protein, comprising: a) transducing a vector containing a gene encoding the FAF1 protein and a promoter regulating the expression of said gene into cells selected from the group consisting of immune cells, stem cells, somatic cells, plant cells, bacterial cells, yeast cells, mammalian cells, and tumor cells to obtain transformed cells; b) culturing the transformed cells; and c) screening the cultured cells for cells that stably express exosomes carrying the FAF1 protein; A method comprising:

17. A method for producing exosomes loaded with FAF1 protein, comprising: a) culturing the cell line according to any one of claims 1 to 15; and b) isolating exosomes loaded with FAF1 protein in cell line medium; A method comprising:

18. 18. The method of claim 17, wherein step b) is performed by size exclusion chromatography, ion exchange chromatography, density gradient centrifugation, differential centrifugation, ultrafiltration, tangential flow filtration, exosome precipitation, total exosome extraction kit, immunoabsorbent capture, affinity capture, affinity purification, immunoassay, microfluidic separation, or a combination thereof.

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