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

Abstract

According to one aspect of the technology disclosed herein, the present invention relates to a cell line in which an FAF1 protein-loaded exosome is stably expressed, and the present invention relates to: a cell line which can produce an FAF1 protein-loaded exosome in a high yield and selectively modulate the expression of an FAF1 protein; a method for preparing a cell line; and a method for producing the exosome loaded with the FAF1 protein.

Description

Technical Field

[0001] The present invention relates to a cell line capable of mass-producing exosomes loaded with FAF1, expressing FAF1 only when necessary and stably expressing exosomes, a method for preparing the cell line, and a method for producing exosomes loaded with FAF1. Background Art

[0002] It is known that tumor cells have resistance to apoptosis due to gene mutations associated with apoptosis. There are many types of signaling pathways and receptors associated with apoptosis, but Fas (also known as Fas receptor, CD95, Apo1 or TNFRSF6) is one of the receptors with excellent apoptosis ability. In addition, it has been reported that the expression level of Fas-associated Factor 1 (FAF1), a protein associated with the Fas pathway, is reduced in a variety of cancers, including lung cancer, colon cancer, liver cancer, prostate cancer, brain cancer and breast cancer (XIE, Feng et al. FAF1phosphorylation by AKT accumulates TGF-Βtype II receptor and drives breast cancer metastasis. Nature communications, 2017, 8.1: 1-16).

[0003] On the other hand, extracellular vesicles refer to nanosized vesicles secreted by cells into the external environment for intercellular information exchange. Extracellular vesicles have various names, such as exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, nanovesicles, outer membrane vesicles, etc., depending on their source, secretion mechanism, size, etc., and contain various substances that exhibit biological activity, such as proteins, lipids, nucleic acids, and metabolites.

[0004] Among extracellular vesicles, exosomes have attracted attention in the field of disease diagnosis and treatment in recent years due to their various advantages. Since exosomes are not living cells, they can be stored and transported for a long time, and since there is no cell division, there is almost no risk of tumor generation. In addition, since exosomes are cell-derived materials, they have little immunogenicity and have the same membrane topology as cells, so drugs / vaccines loaded as cargo materials can be easily delivered to specific cells or tissues. Therefore, exosomes have recently been used to develop drugs for treating tumors. For example, Korean Patent No. 10-2053065 relates to a pH-sensitive exosome composition using hyaluronic acid and doxorubicin, which discloses a method for preparing pH-sensitive exosomes using doxorubicin and a polymer chemically combined with hyaluronic acid and 3-diethylaminopropylamine, and the cancer cell killing effect of the exosomes. In addition, Korean Patent Publication No. 10-2018-0078173 relates to a novel exosome anticancer agent, which discloses a recombinant exosome in which receptor tyrosine kinase and SIRP (i.e., phagocytosis-promoting protein) are presented on its surface, and discloses a recombinant exosome containing asparaginase, protein toxin, a specific antibody against a cancer antigen or a fragment of the antibody, a tumor suppressor gene or an anti-angiogenic factor, etc. as an anticancer protein.

[0005] Since the amount of exosomes naturally secreted by cells is very limited, in order to use exosomes for actual tumor treatment purposes, it is necessary to develop cell lines that produce exosomes in large quantities with high yields.

[0006] On the other hand, when the protein produced by the cell line is a protein that induces apoptosis, the protein will have an adverse effect on the survival and proliferation of the cells, so the productivity of the protein may decrease near the second half of the culture. Therefore, it is necessary to develop a cell line and an exosome production method that can mass-produce exosomes loaded with a protein that induces apoptosis without affecting the survival and proliferation of the cells producing the exosomes. Summary of the invention

[0007] Technical issues

[0008] The object of the present invention is to provide a cell line stably expressing exosomes loaded with FAF1 protein, a method for preparing the cell line, and a method for mass production of exosomes loaded with FAF1 protein using the cell line.

[0009] Problem Solution

[0010] In one aspect of the present invention, the present invention provides a cell line stably expressing exosomes loaded with FAF1 protein.

[0011] In another aspect of the present invention, the present invention provides a method for preparing a cell line stably expressing exosomes loaded with FAF1 protein, the method comprising the following steps: a) transducing a vector into immune cells, stem cells, somatic cells, plant cells, bacterial cells, yeast cells, mammalian cells or tumor cells to obtain transformed cells, wherein the vector comprises a gene encoding FAF1 protein and a promoter regulating the expression of the gene; b) culturing the transformed cells; and c) screening cells stably expressing exosomes loaded with FAF1 protein in the cultured cells.

[0012] In another aspect of the present invention, the present invention provides a method for producing exosomes loaded with FAF1 protein, the method comprising the following steps: a) culturing a cell line stably expressing exosomes loaded with FAF1 protein; and b) isolating exosomes loaded with FAF1 protein from the cell line culture medium.

[0013] Advantageous Effects of the Invention

[0014] According to one embodiment of the present invention, a cell line stably expressing exosomes loaded with FAF1 protein can produce exosomes loaded with FAF1 protein at a high yield, and can produce exosomes loaded with FAF1 protein in large quantities while being less affected by apoptosis induced by FAF1 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown are the results of Western blot showing the results of induction of FAF1 expression with doxycycline in cells in which pTetOne-FAF1 transduction was confirmed.

[0016] Figure 2 is a graph showing the viability of the F5-3 cell line, the F5-4 cell line, and the F3-1J cell line compared to the Expi293F cell line.

[0017] Figure 3 The graph shows the doubling time of F5-3 cell line, F5-4 cell line, and F3-1J cell line compared with Expi293F cell line.

[0018] Figure 4 Figure 2 is a graph showing the expression of exosome-related markers in Expi293F and F5-3 cell lines in response to the presence or absence of doxycycline.

[0019] Figure 5 Shown are the exosome production, FAF1 production, and purity of FAF1 in exosomes of HEK293, Expi293F, and F5-3 cell lines in the presence or absence of doxycycline. DETAILED DESCRIPTION

[0020] The present invention is described in detail below.

[0021] One aspect of the present invention relates to a cell line stably expressing exosomes loaded with FAF1 protein.

[0022] As used herein, the term "FAF1" refers to Fas-associated factor 1, which mediates cell necrosis through JNK-dependent mitochondrial dysfunction and negatively regulates Aurora-A to inhibit the G2 / M phase of the cell cycle, thereby participating in cell proliferation. In addition, FAF1 binds to ubiquitinated proteins and valine-containing proteins (VCPs) and participates in the ubiquitin-proteasome pathway to regulate protein degradation. Unnecessary FAF1 is ubiquitinated by Parkin and degraded through the proteasome pathway.

[0023] FAF1 participates in various biochemical processes, including cell death, inflammation, cell proliferation, and protein homeostasis, 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), which exerts tumor suppressive effects by inhibiting NF-κB and inhibits tumor metastasis through TGF-β signaling.

[0024] As used herein, the term "apoptosis" is a programmed cell death (programmed cell death) that can occur in multicellular organisms. Apoptosis causes cell death due to changes in cell morphology and biochemical changes inside the cell. The process ends with cell swelling and rupture, cell membrane changes, chromatin condensation and chromosome breakage, and cells being engulfed by phagocytes. Compared with necrosis (cell death caused by acute cell damage), apoptosis does not cause harm to the organism, but brings benefits to its life cycle. The formation of fingers and toes during human embryonic differentiation is a typical example of apoptosis. In addition, apoptosis is an important mechanism for cell replacement, tissue remodeling, and damaged cell removal.

[0025] As used herein, the FAF1 protein may include the amino acid sequence of Uniprot ID Q9UNN5, or the amino acid sequence of SEQ ID NO: 1, and preferably, may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0026] As used herein, the term "exosome" refers to a vesicle composed of two layers of phospholipid membranes secreted by cells. Exosomes are known to conduct intercellular signaling, form disease-specific nucleic acids and proteins, and release them into body fluids, and play an important role in intercellular communication by delivering specific nucleic acids, proteins, and lipids that are essential for homeostasis between cells. For example, exosomes are involved in basic physiological processes such as neurotransmission, antigen presentation, immune response, organ development, and reproductive capacity, and are also involved in some pathological diseases, including cancer progression, cardiovascular disease, inflammation, and prion transmission.

[0027] Furthermore, exosomes are secreted into the extracellular environment after late endosomes containing intraluminal vesicles (ILVs), called multivesicular bodies (MVBs), fuse with the cell membrane. MVBs can fuse with the cell membrane to release ILVs, or on the other hand, they can fuse with lysosomes to degrade the contents. The materials present in exosomes are not similar in composition to the cytoplasmic materials, and exosomes can selectively contain RNA and proteins.

[0028] Although there are reports that the production of exosomes is dependent or independent of the endosomal sorting complex required for transport (ESCRT), the exact mechanism is difficult to discover. Cells secrete proteins with signal peptides present in them through the endoplasmic reticulum-Golgi complex. Vesicles containing proteins with signal peptides present in them move to the cell membrane, fuse with the cell membrane, and send the proteins out of the cell. However, proteins without signal peptides may be secreted through an alternative non-classical secretory pathway. When proteins without signal peptides are secreted, the proteins are secreted without or with vesicles. Although the exact mechanism of the non-vesicular secretory pathway is unclear, some proteins are secreted via membrane pores and ATP-binding cassette transporters. Vesicular secretion is carried out via extracellular vesicles (including exosomes) and via vesicles of various sizes.

[0029] The average diameter of the exosomes may be 50 nm to 300 nm, but the present invention is not limited thereto.

[0030] In addition, in another aspect of the present invention, the cell line stably expressing exosomes loaded with FAF1 protein can be a cell line transformed with a vector comprising a gene encoding FAF1 protein and a promoter regulating the expression of the gene. In this case, the FAF1 protein is the same as described above.

[0031] In addition, during the transformation of the cell line stably expressing exosomes loaded with FAF1 protein, a vector containing a linear puromycin marker gene can be used. The vector containing the linear puromycin marker gene can include a puromycin marker gene, an SV40 promoter and an SV40 polyadenylation signal, and can preferably be Tet-One from Takara Bio Inc. (Japan). TM The linear puromycin marker (Cat. No. 631626) is included in the Inducible Expression System.

[0032] As used herein, the term "transformation" refers to a molecular biology technique in which a vector containing a foreign gene different from the original cell is introduced into a cell, where the foreign gene combines with DNA present in the original cell, thereby changing the genetic characteristics of the cell.

[0033] As used herein, the term "vector" refers to a device for expressing a target gene in a host cell. For example, a vector may be a chromosomal vector, an episomal vector, a plasmid vector, a single-stranded or double-stranded RNA or DNA viral vector. In addition, vectors include phagemid vectors, cosmid vectors, phage vectors and viral vectors, such as adenoviral vectors, retroviral vectors and adeno-associated viral vectors. Vectors that can be used as vectors can be produced by engineering plasmids (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.) commonly used in the art, and preferably may be PRSET A. Furthermore, the vector can be introduced into cells by well-known techniques for introducing DNA and RNA into cells.

[0034] In addition, in one aspect of the present invention, the promoter regulating the expression of the gene encoding the protein can be a promoter operated by a protein synthesis inhibitor. The protein synthesis inhibitor can be selected from (Aminoglycoside) antibiotics, Tetracyclines 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, Sodium fluoride, fluoride), Sparsomycin and Trichoderma, but not limited thereto, any protein synthesis inhibitor with a similar mechanism may be substituted.

[0035] Tetracycline antibiotics may include antibiotics selected from doxycycline, minocycline, sarecycline, eracycline, chlortetracycline, demeclocycline, lymecycline, rolicycline, omadacycline, mephedrone, oxytetracycline, tetracycline and tigecycline, and any tetracycline-based antibiotic drugs known in the art to which the present invention belongs may be substituted.

[0036] In another aspect of the 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., an ecdysone-responsive promoter, such as the promoter described in US 6,379,945), a metallothionein promoter (US 6,410,828), and a pathogenesis-related (PR) promoter (US 5,689,044) that can react to chemicals such as salicylic acid, ethylene, thiamine and / or BTH, or a combination thereof.

[0037] In addition, the inducible promoter may be a light-inducible promoter, a metal-inducible promoter, a temperature (low temperature or high temperature)-inducible promoter, a nitrogen deficiency-inducible promoter or an antibiotic-inducible promoter, and may be a promoter known in the art to which the present invention belongs that induces gene expression according to the presence or absence of light, metal, temperature change, nitrogen deficiency, antibiotics, etc. Among them, as antibiotic-inducible promoters, thiostrepton-inducible promoters, tetracycline response element (tetracycline response element, TRE) promoters, Plial promoters, etc. are known, and preferably, the antibiotic-inducible promoter may be a TRE promoter. In addition, the TRE promoter is a promoter activated by the above-mentioned tetracycline antibiotics.

[0038] In another aspect of the present invention, the cell line stably expressing exosomes loaded with FAF1 protein can be a cell line that induces the expression of the gene encoding FAF1 protein under an environment where the promoter regulating the expression of the gene encoding FAF1 protein is activated. In addition, the cell line stably expressing exosomes loaded with FAF1 protein can be a cell line that induces the expression of the gene encoding FAF1 protein in the presence of a protein synthesis inhibitor.

[0039] In one embodiment of the present invention, 1×10 cells to be transformed can be treated with 1 to 10 μg of pTetOne-FAF1 plasmid (TakaraBio Inc., Japan). 5 cells to 1×10 7 Preferably, 1×10 cells to be transformed can be cultured by treating with 5 μg of pTetOne-FAF1 plasmid. 6 However, the present invention is not limited thereto.

[0040] In another aspect 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.

[0041] The immune cells may be selected from dendritic cells, natural killer cells, T cells, B cells, regulatory T cells (Treg cells), natural killer T cells, innate lymphocytes, macrophages, granulocytes, chimeric antigen receptor-T (CAR-T) cells, lymphokine-activated killer (LAK) cells and cytokine-induced killer (CIK) cells, but are not limited thereto.

[0042] Stem cells can be mesoderm stem cells, pluripotent stem cells, multipotent stem cells or unipotent stem cells, but are not limited thereto.Pluripotent stem cells can be embryonic stem cells (ES cells), embryonic germ cells (EG cells) or induced pluripotent stem cells (induced pluripotent stem cells, iPSC), but are not limited thereto.Multipotent stem cells can be adult stem cells, such as mesenchymal stem cells (derived from fat, bone marrow, cord blood or umbilical cord, etc.), hematopoietic stem cells (derived from bone marrow or peripheral blood), neural stem cells, reproductive stem cells, etc., but are not limited thereto. Mesenchymal stem cells (MSC) can be human embryonic stem cell-derived mesenchymal stem cells (hES-MSC), bone marrow-derived mesenchymal stem cells (BM-MSC), umbilical cord-derived mesenchymal stem cells (UC-MSC) or adipose-derived mesenchymal stem cells-conditioned medium (ADSC), but are not limited thereto.

[0043] In addition, stem cells can be autologous or allogeneic stem cells, can be derived from any type of animal, including humans and non-human mammals, can be embryonic stem cells, adult stem cells or induced pluripotent stem cells, but are not limited thereto. The term "embryonic stem cell" refers to cells extracted during embryonic development, which are obtained by extracting the inner cell mass from the blastocyst before the fertilized egg is about to be implanted in the mother's uterus, and culturing the inner cell mass in vitro. Embryonic stem cells refer to pluripotent or totipotent cells that have the ability to self-renew and can differentiate into all tissue cells of an individual, and in a broad sense include embryoid bodies derived from embryonic stem cells.

[0044] The somatic cell may be selected from the group consisting of fibroblasts, chondrocytes, synovial cells, keratinocytes, adipocytes, osteoblasts, osteoclasts and peripheral blood mononuclear cells.

[0045] Tumor cells can be derived from human ovarian cancer cell lines (SKOV3 and OVCAR3), human breast cancer cell lines (MCF-7, T47D, BT-474 and MDA-MB-231), human liver cancer 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), but are not limited thereto.

[0046] As the plant cell line, bacterial cell line or yeast cell line, a cell line known in the art to which the present invention pertains and suitable for protein production can be used.

[0047] In another aspect of the present invention, the cell line expressing the exosomes loaded with FAF1 protein can be derived from a mammalian cell line. The mammalian cell line can be selected from a CHO cell line, a NS0 cell line, a Sp2 / 0 cell line, a BHK cell line, a C127 cell line, a HEK293 cell line, a HEK293T cell line, a HEK-293STF cell line, a 293T / 17 cell line, a 293T / 17SF cell line, a HEK-293.2sus cell line, a HEK-293F cell line, a HT-1080 cell line, a PER.C6 cell line, a NuLi-1 cell line, an AR PE-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, but not limited thereto. Preferably, the cell line expressing exosomes loaded with FAF1 protein can be derived from Expi293F cells, which are suspension cells.

[0048] In another aspect of the present invention, a cell line stably expressing exosomes loaded with FAF1 protein can increase the number of exosomes, the FAF1 protein loaded by exosomes, and the ratio of FAF1 in the protein loaded by exosomes.

[0049] In one aspect of the present invention, the cell line stably expressing 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 where a promoter regulating the expression of a gene encoding FAF1 protein is activated is increased compared to the amount of exosomes loaded with FAF1 protein obtained by cell culture in an environment where the promoter is inactivated.

[0050] In addition, the cell line stably expressing exosomes loaded with FAF1 protein can be a cell line in which the amount of exosomes loaded with FAF1 protein obtained by cell culture under an environment in which the promoter regulating the expression of the gene encoding FAF1 protein is activated is increased by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 11 times or more, 12 times or more, 13 times or more, 14 times or more, or 15 times or more. Preferably, the cell line stably expressing exosomes loaded with FAF1 protein can be a cell line in which the amount of exosomes loaded with FAF1 protein obtained by cell culture under an environment in which the promoter regulating the expression of the gene encoding FAF1 protein is activated is increased by 3 times or more, 5 times or more, 6 times or more, or 12 times or more compared to the amount of exosomes loaded with FAF1 protein obtained by cell culture under an environment in which the promoter is inactivated.

[0051] In another aspect of the present invention, the 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 the protein synthesis inhibitor.

[0052] Furthermore, the cell line stably expressing exosomes loaded with FAF1 protein may 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 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 11 times or more, 12 times or more, 13 times or more, 14 times or more, or 15 times or more compared to the amount of exosomes loaded with FAF1 protein obtained by cell culture in the absence of the protein synthesis inhibitor.

[0053] In another aspect of the present invention, the cell line stably expressing exosomes loaded with FAF1 protein can be a cell line in which the proportion of FAF1 in the protein in the exosomes is increased compared to the HEK293 cell line transformed with a vector comprising a gene encoding the FAF1 protein. In addition, the cell line stably expressing exosomes loaded with FAF1 protein can be a cell line in which the proportion of FAF1 in the protein in the exosomes is increased by 2 times or more, 3 times or more, 4 times or more or 5 times or more, preferably 3 times or more, compared to the HEK293 cell line transformed with a vector comprising a gene encoding the FAF1 protein.

[0054] In another aspect of the present invention, the cell line stably expressing exosomes loaded with FAF1 protein can be a cell line in which the number of exosomes loaded with FAF1 protein is increased, the proportion of FAF1 in proteins in exosomes is increased, or both are increased, compared with a HEK293 cell line transformed with a vector comprising a gene encoding FAF1 protein.

[0055] In one aspect of the present invention, the present invention relates to exosomes obtained from a cell line stably expressing exosomes loaded with FAF1 protein.

[0056] Exosomes can be obtained by common methods for isolating exosomes in the art. Exosomes can be extracted by size exclusion chromatography, ion exchange chromatography, density gradient centrifugation, differential centrifugation, ultrafiltration, tangential flow filtration, exosome precipitation, total exosome extraction kit, immune-absorbent capture, affinity, such as affinity capture or affinity purification, immunoassay, microfluidic separation, or a combination of the above methods.

[0057] According to a specific embodiment of the present invention, the culture medium of the cell line stably expressing the exosomes loaded with FAF1 protein can be collected, centrifuged at 300g for 10 minutes, 2000g for 10 minutes, and 10000g for 30 minutes to separate the supernatant, the supernatant can be filtered using a 0.2μm filter, and can be ultracentrifuged at 150000g for 70 minutes. In addition, the supernatant obtained by centrifugation can be removed, the precipitate is washed by adding PBS, and then centrifuged again at 150000g for 70 minutes, and the supernatant is removed to separate the remaining exosomes in the lower layer, but the present disclosure is not limited thereto.

[0058] In the present invention, the exosomes obtained from the cell line stably expressing the exosomes loaded with FAF1 protein can be stored at -20°C to -80°C.

[0059] In addition, in the present invention, FAF1 protein can be secreted into the extracellular space via exosomes. The FAF1 protein secreted into the extracellular space can induce apoptosis of other cells.

[0060] In one aspect of the present invention, the present invention can provide a method for preparing a cell line stably expressing exosomes loaded with FAF1 protein, the method comprising the following steps: a) transducing a vector into a cell to obtain a transformed cell, the vector comprising a gene encoding FAF1 protein and a promoter regulating the expression of the gene, the cell being selected from immune cells, stem cells, somatic cells, plant cells, bacterial cells, yeast cells, mammalian cells or tumor cells; b) culturing the transformed cells; and c) screening cells stably expressing exosomes loaded with FAF1 protein in the cultured cells. The immune cells, stem cells, somatic cells, tumor cells, FAF1 protein, promoter regulating the expression of the gene encoding FAF1 protein, vector and transformation are the same as above. In addition, 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.

[0061] As used herein, the term "culture" 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 well known in the art.

[0062] Furthermore, the medium that can be used for culturing cells in the present invention refers to a known medium for culturing cells. Examples of the culture medium may include a culture medium selected from commercially produced culture 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), Isocove's Modified Dulbecco's Medium (IMDM), KnockOut DMEM, Essential 8 Medium (E8), or Expi293F expression medium, or artificially synthesized media, or serum-free media, protein-free media, and chemically defined media are commercially available, but are not limited thereto. In addition, the culture medium may contain the above-mentioned protein synthesis inhibitor.

[0063] In another aspect of the present invention, a method for preparing exosomes loaded with FAF1 protein may include culturing a cell line stably expressing exosomes loaded with FAF1 protein, and isolating exosomes loaded with FAF1 protein from the cell line culture medium. The above separation method can be used to isolate exosomes, and preferably, the separation of exosomes can be performed using a centrifuge.

[0064] The expression level of the protein can be used as a marker for selecting the cell line used in the present invention, and the expression of the protein can be measured using methods known in the art. For example, antibodies that specifically detect proteins can be purchased commercially, which can be used to detect proteins by using Western blot, co-immunoprecipitation, immunofluorescence, enzyme-linked immunosorbent assay, etc. By using such antibodies, the expression level of a specific protein can be specifically identified, but the present invention is not limited thereto.

[0065] The present invention will be described in detail below with reference to the following examples.

[0066] However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

[0067] Invention Mode

[0068] <Example 1> Preparation of a cell line capable of producing FAF1 exosomes at high yield

[0069] Example 1.1. Preparation of a FAF1 exosome-stable expression cell line derived from the Expi293F cell line

[0070] [Cell culture] Expi293F cell line (Gibco, USA) was purchased as a base cell for preparing a cell line stably producing exosomes loaded with FAF1 protein (hereinafter referred to as FAF1 exosome stable production cell line). It is known that cells of the Expi293F cell line (hereinafter referred to as Expi293F cells) are suspension cells derived from the HEK 293 cell line, which is derived from human embryonic kidney, can be cultured to high density, is highly transducible, and has a higher protein yield than HEK 293 cells (Thermo Fisher Scientific Expi293F cell line product description, https: / / www.thermofisher.com / order / catalog / product / A14527). In addition, Expi293F cells can be cultured in Expi293 Expression Medium (Expi293 Expression Medium, Gibco, USA), which is a synthetic medium that does not contain serum and external proteins, and is used to exclude exosomes or external proteins contained in serum when producing exosomes (Thermo Fisher Scientific Expi293 Expression Medium product description, https: / / www.thermofisher.com / order / catalog / product / A1435101).

[0071] Expi293F cells were cultured at 3 × 10 5 cells / mL or 4×10 5 The cells were inoculated into Expi293 expression medium (Gibco, USA) in a conical flask at a concentration of 3×10 cells / mL and suspension cells were cultured until the cell concentration reached 3×10 6 In this case, Expi293F cells were maintained by subculturing every 3-4 days with Expi293 expression medium at 8% CO2, 37°C and 125 rpm.

[0072] For transduction, use suspension cells adapted to adherent cells. 6Expi293F cells were seeded in 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 antibiotics-antimycotics (penicillin / streptomycin, Gibco, USA) at 37°C and 5% CO2. After at least 24 hours, it was confirmed that the cells adhered to the culture dish and that the cell morphology changed (adaptation of suspension cells to adherent cells).

[0073] Meanwhile, adapted Expi293F cells were maintained in DMEM medium containing antibiotics, 10% FBS (Atlas Biologicals, USA) and 1% antibiotic-antimycotic (penicillin / streptomycin, Gibco, USA) by subculturing every 3-4 days at 5% CO2 and 37°C.

[0074] 1×10 cells adapted to adherent cells 6 Expi293F cells were seeded in a 100 mm culture dish and 3 × 10 5 Expi293F cells were seeded in 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% CO2 for 24 hours.

[0075] [Transduction] The pTetOne-FAF1 plasmid was prepared by using the pTetOne plasmid (TaKaRa Bio, Japan) and the FAF1 amino acid sequence of SEQ ID NO: 1, and the pTetOne plasmid (TaKaRa Bio, Japan) without FAF1 was used as a control. The pTetOne-FAF1 plasmid / control plasmid (3 μg or 5 μg) and the 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, and the resulting mixture was reacted 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, and the resulting mixture was reacted at room temperature for 5 minutes. The plasmid-puromycin linear marker-medium mixture was mixed with the ExpiFectamine-medium mixture and reacted at room temperature for 10 minutes.

[0076] The reaction mixture was added to Expi293F cells cultured in DMEM medium in a culture dish for 24 hours under the conditions of 5% CO 2 and 37° C. to transduce the pTetOne-FAF1 plasmid into the cells.

[0077] Example 1.2. Screening of cell lines that stably produce FAF1 exosomes

[0078] The cells transduced with the pTetOne-FAF1 plasmid prepared in Example 1.1 were cultured in the same culture dish, and the DMEM medium containing 10% FBS and puromycin was replaced every 2-3 days. Considering the rate of cell apoptosis, puromycin was added to the culture medium at a concentration of 1 μg / mL or 3 μg / mL.

[0079] One to two weeks after pTetOne-FAF1 plasmid transduction, non-transduced cells without puromycin resistance were killed. When the transduced cells with puromycin resistance formed colonies, the colonies were isolated using a Cloning cylinder (Sigma-Aldrich, USA) and cultured in 35 mm culture dishes to obtain 13 cell lines with puromycin resistance (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).

[0080] Then, to investigate whether FAF1 was expressed in the 13 cell lines with puromycin resistance transduced by the TetOne system, doxycycline was added, and the expression level of FAF1 was measured by western blotting.

[0081] 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 cultured at 3 × 10 5 Inoculate in 2 60mm culture dishes, then culture in 10% FBS DMEM medium containing or not containing 100ng / mL doxycycline for 3 days. Considering the half-life (1 day) of doxycycline in the medium, the medium was changed once a day. After 3 days, the cultured cells were collected and the expression level of FAF1 in the cells treated with doxycycline was compared with that in the cells not treated with doxycycline by Western blotting.

[0082] In order 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 culture medium were centrifuged at 300g for 10 minutes, and then the supernatant was removed to obtain a cell pellet. Mammalian lysis buffer (80 μL) was added to the cell pellet, and the cells were dissolved on ice for 30 minutes. The protein in the cell pellet was diluted with Bradford solution, and then its concentration was measured at a wavelength of 595nm on a spectrophotometer (MECASYS, South Korea). A certain amount of precipitation was dissolved to obtain 20 μg of protein, mixed with 5 μL of SDS sample buffer, and reacted in boiling water for 3 minutes. A 10% acrylamide gel was made using a Western blotting kit (Amersham Biosciences, UK), and a certain amount of cell protein samples boiled for 3 minutes were placed on the gel, and the protein was separated by size by applying a voltage of 80 to 100V. The acrylamide gel containing the separated protein was overlapped with a nitrocellulose membrane (GE Healthcare, the United States), and a 200 mA current was applied thereto for 2 hours to transfer the protein to the nitrocellulose membrane. The nitrocellulose membrane to which the protein was transferred was blocked with skim milk, anti-FAF1 antibodies (E-4, Santacruz, 1:1000, mouse IgGs) were attached, and then secondary antibodies (HRP-anti-mouse, 31439, Thermo Fisher Scientific, 1:1000, goat IgGs) were attached, and the amount of FAF1 protein was confirmed by Western blotting detection kit (AbFrontier, South Korea) and ChemiDoc-It Imaging System (UVP, the United States), and the Western blotting results were quantified by ImageJ. Β-actin and GAPDH were used as loading controls.

[0083] Cells with a 3-fold or greater increase in FAF1 production compared to cells not treated with doxycycline were used as the screening criteria for stable FAF1 exosome-producing cell lines. Three cell lines (F5-3, F5-4, and F3-1J) met the screening criteria for stable FAF1 exosome-producing cell lines ( Figure 1 ). Compared with 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 F5-3, F5-4, and F3-1J cell lines, 1×10 cells were maintained by subculturing every 3–4 days in an Erlenmeyer flask for cell culture at 8% CO2, 37°C, and 125 rpm in Expi293 expression medium (Gibco, USA). 7cells and adapted them into suspension cells, thereby establishing a stable FAF1 exosome production cell line that can be turned on / off.

[0084] Example 1.3. Cell viability and doubling time of FAF1 exosome stably expressing cell lines that can be turned on / off

[0085] The FAF1 exosome stably expressing cell lines F5-3, F5-4, and F3-1J that can be turned on / off were subcultured under the conditions of Example 1.2 above. In these FAF1 exosome stably expressing cell lines, when the cell division rate was slow, the cell concentration inoculated into the conical flask for cell culture was set to 6×10 5 cells / mL or 8×10 5 cells / mL, so the culture was carried out under the conditions of Example 1.2 so that the cell concentration reached 3×10 within 3-4 days. 6 The concentration of cells / mL.

[0086] The Expi293F cell line was maintained by subculturing every 3-4 days in Expi293 expression medium (Gibco, USA) at 8% CO2, 37°C and 125 rpm.

[0087] The cell culture medium (10 μL) was separated and mixed with 140 μL Muse Count & Viability Kit (Luminex, USA) and reacted at room temperature for 5 minutes. Then, the mixture was added to the Muse cell analyzer (Luminex, USA) to measure the cell number and viability.

[0088] Using the cell numbers measured during subculture, calculate the doubling time using the following formula.

[0089]

[0090] T = incubation time

[0091] C = cell concentration after culture

[0092] C0 = cell concentration before culture

[0093] During at least 10 subcultures of Expi293F cells, F5-3 cells, F5-4 cells, and F3-1J cells, viability and cell concentration were measured by Muse Cell Analyzer ( Figure 2 and Figure 3). The average viability of each cell was measured, and the average viability of Expi293F cells was 95.4±1.12%, F5-3 cells was 90.8±2.63%, F5-4 cells was 92.0±2.64%, and F3-1J cells was 90.7±4.53%. The viability of the prepared FAF1 exosome stable expression cell line was slightly decreased compared with the Expi293F cell line, but all cell lines showed good viability of 90% or higher.

[0094] The doubling time of each cell was calculated using the measured cell concentration, which was 24.4±1.75 hours for Expi293F cells, 33.8±4.76 hours for F5-3 cells, 27.7±3.40 hours for F5-4 cells, and 30.4±4.17 hours for F3-1J cells. The doubling time of the prepared FAF1 exosome stably expressing cell line was slightly increased compared with the Expi293F cell line. It is speculated that the lower viability and longer doubling time compared with the original Expi293F cell line are because the transduced TetOne-FAF1 system does not completely inhibit the expression of FAF1 that induces apoptosis.

[0095] <Example 2> Characterization of exosomes from a cell line stably expressing FAF1 exosomes Example 2.1. Isolation of exosomes using an ultracentrifuge

[0096] Exosomes from cell lines stably expressing FAF1 and Expi293F cell line were isolated using an ultracentrifuge (Optima XE-100, BECKMAN Coulter, USA).

[0097] Specifically, the concentration was 3×10 6 Expi293F, F5-3, F5-4, and F3-1J cells at 1 μg / mL were cultured in 50 mL of Expi293 expression medium containing 1 μg / mL doxycycline for 48 or 72 hours without changing the medium to isolate all exosomes during the culture period. The culture medium of 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 using a 0.2 μm syringe filter (BioFACT, Korea).

[0098] To isolate the exosomes used in the experiment, 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 precipitate 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.

[0099] The isolated exosomes were stored at -80°C and used within one week after isolation or thawed and stored at 4°C for future use.

[0100] Example 2.2. Confirmation of cells stably expressing FAF1 exosomes and the loading of FAF1 and exosome-related markers in isolated exosomes

[0101] In order to investigate whether the exosomes isolated from the FAF1 exosome stable expression cell line of Example 1.2 are the same as the exosomes isolated from the Expi293F cell line as the parent cell, the protein amounts of Expi293F cells and exosomes isolated therefrom were compared with those of F5-3 cells and exosomes isolated therefrom, which had the highest FAF1 expression level in the protein blotting experiment of Example 1.3, by Western blotting.

[0102] Specifically, cells and exosomes were separated by the method of Example 2.1, respectively. In the control group, Expi293F cells and F5-3 cells were cultured for 48 hours without doxycycline, and in the experimental group, the cells were treated with 1 μg / mL doxycycline and cultured for 48 hours or 72 hours.

[0103] For Western blotting, the cell culture medium was centrifuged at 300 g for 10 minutes to obtain a cell pellet, and the supernatant was removed. Mammalian lysis buffer (50 μL) was added to the cell pellet, and the cell pellet was lysed on ice for 30 minutes.

[0104] The dissolved precipitate was mixed with 5 μL of SDS sample buffer and reacted 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 reacted in boiling water for 3 minutes. 8-10% acrylamide gel was prepared using a Western blotting kit (Amersham Biosciences, UK). In order to perform Western blotting of equal amounts of protein, the intracellular protein was diluted with Bradford solution and the concentration was measured at a wavelength of 595 nm on a spectrophotometer (MECASYS, South Korea).

[0105] Nanoparticle tracking analysis (NTA) was used to determine the concentration of exosomes. The isolated exosomes were diluted to a concentration of 1×10 8 particles / mL to 1×10 9 The concentration of exosomes was determined using NS300 (Malvern Panalytical, UK), and 5×10 9Exosomes were placed on an acrylamide gel. After boiling for 3 minutes, cells and exosome protein samples were placed on the gel in a certain amount, and a voltage of 80-100V was applied to separate proteins by size. The acrylamide gel containing the separated proteins was overlapped with a nitrocellulose membrane, and a 200mA current was applied for 2 hours to transfer the proteins to the nitrocellulose membrane. The nitrocellulose membrane to which the proteins were transferred was blocked with skim milk, and the primary antibodies in Table 1 below were attached to it, and then the secondary antibodies in Table 1 below were attached. The amount of FAF1 and exosome-related marker proteins (Alix, CD81, CD47, CD9, and Syntenin-1) was determined by a Western blotting detection kit (AbFrontier, South Korea) and a ChemiDoc-It imaging system (UVP, USA), and GAPDH was used as a cell loading control.

[0106] [Table 1]

[0107]

[0108]

[0109] Cellular protein (10 or 20 μg) was added according to the type of antibody used for Western blotting. When the amount of FAF1 loaded in exosomes was quantified by Western blotting, a predetermined amount of recombinant human FAF1 protein (BOSTER, USA) was subjected to Western blotting together and measured using ImageJ, and the measured value was compared with the measured value of FAF1 in exosomes.

[0110] Only when F5-3 cells were treated with doxycycline did intracellular FAF1 increase significantly. When F5-3 cells were treated with doxycycline, the amount of FAF1 loaded in exosomes also increased significantly ( Figure 4 , F5-3 cells and F5-3 Exo). In particular, the loading amount of FAF1 was the largest when the cells were cultured for 72 hours. All exosome-related markers were present in exosomes, thus it can be determined that the protein is a protein in exosomes.

[0111] Example 2.3. Comparison of the number of exosomes between HEK293 cell line and FAF1 exosome stable expression cell line, and the amount of FAF1 loaded

[0112] The differences in FAF1-loaded exosome production were compared between HEK293 cells transduced with 3x Flag tag-FAF1 plasmid / control plasmid and cultured for 24 hours, Expi293F cells cultured in the absence of doxycycline for 48 hours, Expi293F cells treated with 1 μg / mL doxycycline and cultured for 48 hours, F5-3 cells cultured in the absence of doxycycline for 48 hours, and F5-3 cells treated with 1 μg / mL doxycycline and cultured for 48 hours.

[0113] HEK293 cells were maintained in DMEM medium containing 10% FBS and antibiotics by subculturing every 3-4 days at 5% CO2 and 37°C.

[0114] To transiently overexpress FAF1 in HEK293 cells, 3x Flag tag-FAF1 plasmid / control plasmid (8 μg) were transduced into 6.5 × 10 6 HEK293 cells. Specifically, HEK293 cells were inoculated into 150 mm culture dishes and then cultured in DMEM medium containing 10% FBS at 5% CO2 and 37°C for 24 hours. 3x Flag tag-FAF1 was prepared by a previously known method (Yu et al., FAF1 mediates regulated necrosis through PARP1 activation uponoxidative stress leading to dopaminergic neurodegeneration. Cell Death&Differentiation, 2016, 23.11: 1873-1885). DNA: BioT (Biolandscientific, USA) (ratio of 1: 1.5) was mixed in DMEM medium without FBS and antibiotics to form a total of 180 μL of mixture, and the mixture was reacted at room temperature for 5 minutes. The mixture was added to cells cultured for 24 hours and reacted for 24 hours at 5% CO2 and 37°C.

[0115] To isolate exosomes, the culture medium of the transduced HEK293 cells was replaced with DMEM medium without FBS and antibiotics, and the cells were cultured for 24 hours. When HEK293 cells transiently overexpressing FAF1 are cultured for a long time without FBS, apoptosis occurs, so the cells were cultured for only 24 hours, and exosomes were isolated by the method of Example 2.1.

[0116] Expi293F cells and F5-3 cells were cultured in Expi293 expression medium or in Expi293 expression medium containing 1 μg / mL doxycycline.

[0117] The amount of FAF1 loaded in exosomes isolated from HEK293, Expi293F, and F5-3 cells was quantified by Western blotting and ELISA.

[0118] When exosomes were quantified by the Western blotting method of Example 2.2, the recombinant FAF1 protein whose protein amount was accurately determined was loaded together, the protein bands were determined by ImageJ, and then compared and quantified.

[0119] ELISA analysis was performed using a human FAF1 ELISA kit (Abbexa, UK). Mammalian lysis buffer (10 μL) was added to exosomes isolated from HEK293, Expi293F, and F5-3 cells, respectively, and the exosomes were lysed on ice for 30 min. Then, the exosomes were diluted with standard / sample diluent to allow 1 × 10 per well of the 96-well plate of the ELISA kit to be added. 8 or 1×10 7 HEK293 exosomes, 1×10 9 or 1×10 8 Expi293F and F5-3 exosomes. Dilute the FAF1 standard to a concentration of 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 ng / mL, and inoculate 100 μL of the FAF1 standard and the diluted exosomes into the 96-well plate of the ELISA kit, and react at 37°C for 2 hours. Discard the supernatant, dilute the A solution in the ELISA kit into a total solution, inoculate 100 μL each into it, and react at 37°C for 1 hour. Discard the supernatant, then add the wash buffer and wash twice. Discard the supernatant, dilute the B solution in the ELISA kit into a solution of the B solution, inoculate 100 μL each into it, and react at 37°C for 1 hour. Discard the supernatant, then add the wash buffer and wash three times. Discard the supernatant, then inoculate 90 μL each of the TMB solution into it, react at 37°C for 20-30 minutes, and inoculate 50 μL each of the stop solution into it. Each well was measured at 450 nm with a microplate reader (PerkinElmer, USA) (the color of the well changed according to the amount of protein) to quantify FAF1 protein. The average value was obtained by combining the measured values, which were the amount of loaded FAF1 and the number of exosomes measured by Western blotting and ELISA.

[0120] In addition, the proportion of FAF1 in the total protein of one exosome was determined. Exosome proteins were dissolved with mammalian lysis buffer, exposed to exosomes, and then diluted with Bradford solution, and the amount of protein was measured at a wavelength of 595 nm using a spectrophotometer (MECASYS, Korea).

[0121] Figure 5 The amount of FAF1 loaded, the measurements of the number of exosomes, and the proportion of FAF1 to the total protein in a single exosome are shown.

[0122] For the number of exosomes produced based on 50 mL of culture medium, Expi293F and F5-3 cells were at least 3.7 times that of HEK293 cells. This means that when using the same culture medium, Expi293F and F5-3 cells can be cultured more densely and thus produce more exosomes at one time. When comparing the exosome yields of FAF1-loaded exosomes, doxycycline-induced F5-3 cells produced 71.2 times more exosomes than HEK293 cells.

[0123] Based on 50mL of culture medium, the total amount of FAF1 protein loaded on exosomes was measured, which was 70ng in HEK293 exosomes and 2287ng in F5-3 exosomes, confirming that F5-3 cells produced 32.7 times more FAF1 protein than HEK293 cells. This shows that although the amount of FAF1 loaded per exosome of F5-3 cells is less than that of HEK293 cells, the total amount of FAF1 that F5-3 cells can produce at one time is 32.7 times that of HEK293 cells when using the same volume of culture medium, and more FAF1 can be delivered to the target.

[0124] The amount of FAF1 protein measured in each cell was compared with the amount of exosome protein, and 0.41% was measured in HEK293 exosomes transduced with 3xFlag tag-FAF1, and 1.43% was measured in F5-3 exosomes treated with 1μg / mL doxycycline and cultured for 48 hours. This suggests that exosomes isolated from HEK293 cells contain more impurities than F5-3, because not only FAF1 protein but also various other proteins are present.

[0125] Therefore, it was confirmed that exosomes isolated from F5-3 cells produced more FAF1 protein and more and purer exosomes when the same volume of culture medium was used.

Claims

1. A cell line stably expressing exosomes loaded with FAF1 protein. 2 . The cell line according to claim 1 , wherein the cell line is transformed with a vector comprising a gene encoding FAF1 protein and a promoter regulating the expression of the gene. 3 . The cell line according to claim 2 , wherein the expression of the gene encoding the FAF1 protein is induced in an environment where a promoter regulating the expression of the gene is activated. The cell line according to claim 2 , wherein the FAF1 protein comprises the amino acid sequence of SEQ ID NO:

1. The cell line according to claim 2 , wherein the promoter regulating the expression of the gene encoding the FAF1 protein is controlled by a protein synthesis inhibitor.

6. The cell line according to claim 5, wherein the protein synthesis inhibitor is selected from the group consisting of aminoglycoside antibiotics, tetracycline antibiotics, macrolide antibiotics, lincosamide antibiotics, streptogramin antibiotics, pleuromutilin antibiotics, phenicol antibiotics, fusidic acid antibiotics, oxazolidinone antibiotics, anisomycin antibiotics, edinamycin antibiotics, pactamycin antibiotics, puromycin antibiotics, cyclohexamide, aurintricarboxylic acid, diphtheria toxin, ricin, sodium fluoride, fluoride), Sparsomycin and Trichoderma.

7. The cell line of claim 6, wherein the tetracycline antibiotic is selected from the group consisting of doxycycline, minocycline, sarecycline, eracycline, chlortetracycline, demeclocycline, lymecycline, rolicycline, omadacycline, mephedrone, oxytetracycline, tetracycline and tigecycline. 8 . The cell line according to claim 5 , wherein the promoter regulating the expression of the gene encoding the FAF1 protein is a tetracycline response element (TRE) promoter.

9. The cell line according to claim 2, wherein a vector containing a linear puromycin marker gene is also used in the transformation process. 10 . The cell line according to claim 1 , which is derived from immune cells, stem cells, somatic cells, mammalian cell lines or tumor cells.

11. The cell line according to claim 10, wherein the mammalian cell line is selected from the group consisting of CHO cell line, NS0 cell line, Sp2 / 0 cell line, BHK cell line, C127 cell line, HEK293 cell line, HEK293T cell line, HEK-293STF cell line, 293T / 17 cell line, 293T / 17SF cell line, HEK-293.2sus cell line, HEK-293F cell line, 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, WT Cell lines of 9-7 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 according to claim 10, wherein the mammalian cell line is Expi293F cell line.

13. The cell line according to claim 2, wherein the amount of exosomes loaded with FAF1 protein obtained by cell culture under an environment in which a promoter regulating expression of a gene encoding FAF1 protein is activated is increased by 2 to 15 times compared to the amount of exosomes loaded with FAF1 protein obtained by cell culture under an environment in which the promoter is inactivated. The cell line according to claim 1 , wherein the proportion of FAF1 in the proteins in the exosomes is increased at least 3-fold compared to a HEK293 cell line transformed with a vector comprising a gene encoding the FAF1 protein. 15 . The cell line of claim 1 , wherein the number of exosomes loaded with FAF1 protein is increased, the proportion of FAF1 in proteins in exosomes is increased, or both are increased compared to a HEK293 cell line transformed with a vector comprising a gene encoding FAF1 protein.

16. A method for preparing a cell line stably expressing exosomes loaded with FAF1 protein, the method comprising the following steps: a) transducing a vector into a cell to obtain a transformed cell, wherein the vector comprises a gene encoding a FAF1 protein and a promoter regulating the expression of the gene, and the cell is selected from immune cells, stem cells, somatic cells, plant cells, bacterial cells, yeast cells, mammalian cells or tumor cells; b) culturing the transformed cells; and c) Screening cells that stably express exosomes loaded with FAF1 protein in cultured cells.

17. A method for producing exosomes loaded with FAF1 protein, the method comprising the following steps: a) culturing the cell line according to any one of claims 1 to 15; and b) Exosomes loaded with FAF1 protein were isolated from the culture medium of the cell lines.

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

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