New application of cross-flow filtration device for preparing functional exosomes

Abstract

The present invention relates to a novel use of a cross-flow filtration device for preparing functional exosomes and a continuous concentration system for preparing ultra-high concentrated exosome solutions.

Description

Technical Field

[0001] The present disclosure relates to novel uses of tangential flow filtration devices and continuous concentration systems for producing ultra-high concentrated exosome solutions for medical applications. Background Art

[0002] It is known that various cells present in multicellular organisms (including humans) secrete nano-sized endoplasmic reticulum called "exosomes". Exosomes are vesicles with the same membrane structure as the cell membrane and are known to play a role in delivering membrane components, proteins, RNA, etc. to other cells and tissues. In particular, exosomes secreted from stem cells contain various growth factors and cytokines secreted by stem cells and are known to control behaviors such as cell adhesion, growth, differentiation, etc. In addition, since impurities such as cell waste, antibiotics, serum, etc. in the cell culture solution are removed during the separation process, the exosomes can be used safely while having the same effect as the cell culture solution.

[0003] Exosomes are approximately 100 nm in size and carry out cell-to-cell signaling. Their role as transport mediators for the exchange of proteins, genetic material, and other substances between cells has been revealed, leading to research into using exosomes to control the behavior of tissues (including cells).

[0004] Exosomes are known as substances that maximize the ability to deliver proteins and genetic material derived from parent cells to specific cells. These exosome therapeutics have therapeutic efficacy exceeding the "cytokine effect" of conventional cell therapeutics, delivering therapeutically effective proteins and genetic material to cells surrounding diseased tissues to treat the tissues. Research is ongoing to control or analyze the properties of exosomes by conferring functionality by allowing specific substances to react with the exosome's lipid bilayer or interior.

[0005] Many documents are cited throughout this specification, and their citations are indicated. The disclosures of the cited documents are incorporated into this specification in their entirety by reference to more clearly describe the level of the art to which this disclosure belongs and the content of this disclosure. Summary of the Invention

[0006] Technical issues

[0007] One object of the present disclosure is to provide new uses for conventional tangential flow filtration devices that are limited to concentration, purification, and buffer exchange methods, and to provide methods for preparing functional exosomes using only the tangential flow filtration devices.

[0008] In addition, an object of the present disclosure is to provide a multiplex TFF continuous concentration device for ultra-high concentration of exosomes from a solution containing exosomes (eg, a stem cell culture solution), and a method for producing an ultra-high concentrated exosome solution.

[0009] More specifically, one object of the present disclosure is to provide the following embodiments.

[0010] Embodiment 1. A method for preparing functional exosomes, comprising modifying the exosomes with a functional material using a tangential flow filtration (TFF) apparatus.

[0011] Embodiment 2. The method according to embodiment 1, wherein the functional material is one or more selected from the group consisting of: biocompatible polymers, protein drugs, chemical drugs, and labeling molecules.

[0012] Embodiment 3. A method according to any one of the preceding embodiments, wherein the method comprises the following steps: a reaction step of modifying the exosome with a functional material by mixing the exosome and the functional material in a mixed solution; a filtering and concentration step of removing unreacted functional material, and a washing step of replacing the solvent in the mixed solution, wherein the reaction step, the filtering and concentration step, and the washing step are performed using a tangential flow filtration device (first TFF device).

[0013] Embodiment 4. The method according to any one of the preceding embodiments, wherein the reacting step is performed while the mixed solution of the exosome and the functional material is circulated by a pump of the TFF device.

[0014] Embodiment 5. The method of any one of the preceding embodiments, wherein the method does not use a separate batch reactor to react the exosomes with the functional material.

[0015] Embodiment 6. The method according to any one of the preceding embodiments, wherein the method does not use a separate stirring device and a separate washing device other than the TFF apparatus.

[0016] Embodiment 7. The method according to any one of the preceding embodiments, wherein the method uses the TFF device to simultaneously perform the reacting step, the filtering and concentrating step, and the washing step.

[0017] Embodiment 8. The method according to any one of the preceding embodiments, wherein the method is used to produce the functional exosomes on a large scale.

[0018] Embodiment 9. A method according to any one of the preceding embodiments, wherein the method further comprises the step of injecting the concentrated functional exosomes obtained from the first TFF device into a multiple TFF continuous concentration device to further concentrate the exosomes, wherein the multiple TFF continuous concentration device comprises n TFF devices, and the n TFF devices are connected to form a closed system isolated from the outside, and n concentration processes are continuously performed in the closed system, and wherein n is an integer from 1 to 10.

[0019] Embodiment 10. A method for preparing an ultra-high concentrated exosome solution, comprising: injecting a raw material solution containing exosomes into a multiple TFF continuous concentration device, in which n TFF devices are connected to form a closed system isolated from the outside; and continuously performing n concentration processes in the closed system using the multiple TFF continuous concentration device, wherein n is an integer from 1 to 10.

[0020] Embodiment 11. The method according to any one of the preceding embodiments, wherein the concentration of exosomes in the final ultra-high concentrated exosome solution obtained after the n consecutive concentration processes is 25 to 50% of the concentration of exosomes in the raw material solution. n times higher.

[0021] Embodiment 12. The method according to any one of the preceding embodiments, wherein the concentration of exosomes in the final ultra-high concentrated exosome solution after the n consecutive concentration processes is 10 7 to 10 13 particles / mL.

[0022] Other objects and advantages of the present disclosure will become more apparent from the accompanying detailed description of the invention, the claims and the drawings.

[0023] Technical Solution

[0024] Novel use of tangential flow filtration devices for generating functional exosomes

[0025] One aspect of the present disclosure is to provide a method for producing functional exosomes, characterized in that the exosomes are modified with functional materials using a tangential flow filtration (TFF) device.

[0026] "Functional exosomes" refer to exosomes that are endowed with biochemical functions by loading or cross-linking functional materials such as biochemical molecules within the exosome or inside / outside the bilayer lipid membrane of the exosome, and according to the type and function of the functional material, they can be selectively applied to one or more selected from the following: exosome labeling technology, exosome drug loading technology, exosome therapeutic technology, etc.

[0027] The conventional technology for preparing functional exosomes is through a step-by-step process including: ① reaction of exosomes with functional materials; ② separation and purification of functional exosomes; and ③ complete product generation by additional concentration or dilution.

[0028] During the reaction process, after the exosome and functional material are loaded into a reactor or container, the functional material and exosome are allowed to react under a stirring environment. Subsequently, during the transfer process, the incorporation of exogenous substances can occur. In addition, to create a stirring environment, additional equipment such as a magnetic stirrer or propeller stirring system is required.

[0029] During the separation and purification process, ultracentrifugation or dialysis is used to remove unreacted residues. Ultracentrifugation refers to a method of obtaining pure exosomes with improved properties by diluting a large amount of buffer in the reactants to remove unreacted substances and performing ultracentrifugation. Residual reactants cannot be completely removed in a single process and can be completely removed by repeating washing 3 or more times. Since repeated ultracentrifugation is required, the yield of exosomes may be very low. In addition, a high rotation speed of 150,000×g or higher and a processing time of 1 hour or more per cycle are required. Dialysis refers to a method of removing residual reactants by a dialysis membrane that is smaller than the exosomes and has a greater filtration function than the reactants. It is a method of immersing the exosome reactants contained in the dialysis tubing in a large amount of buffer solution to remove residual reactants outside the dialysis tubing, and a long washing time is required to obtain pure exosomes.

[0030] Therefore, in conventional exosome modification technologies, generally, 1) the reaction process and the separation and purification process, as well as the washing process are not performed simultaneously, and 2) repeated and time-consuming washing processes are required to remove residual reactants.

[0031] In addition, conventional techniques for producing functional exosomes are only suitable for modifying less than 100 mL of exosomes at a time, and if they are scaled up to a commercial level, the process time and cost increase significantly, and exosome losses may occur.

[0032] In this context, the present disclosure provides a method for producing scaled-up exosomes on a commercial level by not only simultaneously performing a reaction process, a separation and purification process, and a washing process of functional exosomes using a tangential flow filtration device, but also producing a large amount of functional exosomes in a short time.

[0033] Generally speaking, a tangential flow filtration device includes 1) a process of circulating a biological solution, 2) a process of removing proteins and impurities smaller in size than the filtration filter, and 3) a process of replacing the solvent of the solution.

[0034] According to one embodiment of the present disclosure, in the process 1), the exosome surface modification is performed while the exosome and the functional material are mixed and reacted. Then, the mixed solution of the exosome and the functional material is circulated by a pump, thereby eliminating the need for a separate stirring process.

[0035] In the process 2), unreacted functional materials that do not participate in the reaction, such as proteins, dyes, polymers, etc., are removed by filtration, and a solution containing reacted exosomes can be obtained. This process may include a process of recycling the solution containing reacted exosomes and concentrating it to a desired concentration.

[0036] In the process of 3), in order to remove residual reaction impurities, the solvent is replaced with a buffer solution or water for injection. For example, the solvent can be one or more selected from the following: phosphate buffered saline (PBS), tris buffered saline (TBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered saline (HBS) physiological saline solution, distilled water, culture medium and water for injection.

[0037] Therefore, the present invention provides a method for producing functional exosomes, characterized in that it includes: a reaction step of mixing exosomes and a functional material; a filtration and concentration step of removing unreacted functional material; and a washing step of replacing the solvent of the exosome solution, and a tangential flow filtration device is used to perform the reaction step, the filtration and concentration step, and the washing step.

[0038] When the surface-modified exosomes are produced through the above-described series of processes, separate batch reactors, stirring equipment, and washing equipment are unnecessary, and additional concentration and washing can be performed in a short time in addition to the reaction time.

[0039] Therefore, the present disclosure provides a novel method for producing functional exosomes using a tangential flow filtration method that can be used for limited applications (including conventional separation and filtration, concentration), and simultaneously performing a reaction step (biochemical modification of exosomes), a filtration and concentration step, and a washing step without the need for separate washing and stirring processes, and thus achieving higher productivity and yield than conventional techniques.

[0040] The tangential flow filtration device used in one embodiment of the present disclosure is an ultrafiltration system, and as a result of performing the reaction step, filtration and concentration steps, and washing steps using a TFF device, the exosome solution can be effectively concentrated by 1 / 10 to 1 / 100 in volume.

[0041] Ultrafiltration (between microfiltration and reverse osmosis) is a method for separating specific substances by utilizing the size difference between membrane pores and solutes. The molecular weight cutoff (MWCO) of an ultrafiltration membrane is defined as the minimum molecular weight of a solute that is 90% or more excluded by the membrane and can demonstrate its separation performance.

[0042] The tangential flow filtration used in the present disclosure may be one or more selected from hollow fiber TFF and membrane TFF capable of ultrafiltration, and preferably, it may use a TFF filter having a molecular weight cutoff (MWCO) of 100,000 to 500,000 Da.

[0043] In the present disclosure, the term "modification" means loading or cross-linking functional materials (eg, biochemical substances) on the surface of the bilayer lipid membrane of the exosome or inside the exosome.

[0044] In one embodiment, the functional material may be one or more selected from the group consisting of: biocompatible polymers, protein drugs, chemical drugs, and labeling molecules.

[0045] The biocompatible polymer can be one or more selected from the group consisting of hyaluronic acid (HA), gelatin, chitosan, collagen, alginic acid, pectin, carrageenan, chondroitin, chondroitin sulfate, dextran, dextran sulfate, polylysine, carboxymethyl chitin, fibrin, agarose, pullulan, cellulose, polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol (PVA), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl cellulose, polyols, gum, gum arabic, alginate, cyclodextrin, dextrin, glucose, fructose, starch, trehalose, glucose, maltose, lactose, lactulose, fructose, turanose, raffinose, melezitose, dextran, sorbitol, xylitol, palatinate polylactic acid, polyglycolic acid, polyethylene oxide, polyacrylic acid, polyacrylamide, polymethacrylic acid, and polymaleic acid.

[0046] The protein drug may be one or more selected from the group consisting of hormones, cytokines, enzymes, antibodies, growth factors, transcriptional regulators, blood factors, vaccines, structural proteins, ligand proteins and receptors, cell surface antigens, receptor antagonists, and toxins.

[0047] The chemical drug may be one or more selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors and anticancer agents (cyclophosphamide, methotrexate, 5-fluorouracil, vinorelbine, Doxorubicin Docetaxel Bleomycin Vinblastine Dacarbazine vincristine Procarbazine prednisolone Etoposide Epirubicin wait).

[0048] The labeling molecule can be one or more selected from the following: fluorophores (fluorescein, phycoerythrin, rhodamine, lissamine and Cy3 and Cy5 (Pharmacia) etc.), chromophores, chemiluminescent groups, magnetic particles, radioisotopes (C 14 , I 125 、P 32 and S 35 etc.), labels, electron-aggregating particles, enzymes (such as alkaline phosphatase and horseradish peroxidase), cofactors, enzyme substrates, heavy metals (such as gold) and antibodies, streptavidin, biotin, digoxigenin and heptane with specific binding partners (such as chelating groups).

[0049] In one embodiment of the present disclosure, the exosome modified by the functional material may be an exosome secreted when primary cells such as stem cells, cancer cells, tissue cells, etc., and cell lines such as stem cell lines, cancer cell lines, etc. are cultured.

[0050] The stem cell line can be one or more selected from the following: an embryonic stem cell line, an adult stem cell line, and an induced pluripotent stem cell (iPSC) line; the cancer cell line can be one or more selected from the following: a bladder cancer cell line, a blood cancer (leukemia, myeloma) cell line, a bone cancer cell line, a brain cancer cell line, a breast cancer cell line, a cervical cancer cell line, a colorectal cancer cell line, an endometrial cancer cell line, an esophageal cancer cell line, a fibrosarcoma cell line, a renal cancer cell line, a liver cancer cell line, a lung cancer cell line, a lymphoma cell line, a neural cancer cell line, an oral cancer cell line, an ovarian cancer cell line, a pancreatic cancer cell line, a prostate cancer cell line, a skin cancer cell line, a spleen cancer cell line, a gastric cancer cell line, a testicular cancer cell line, a thyroid cancer cell line, and a uterine cancer cell line.

[0051] The functional exosome modified by the production method of the present disclosure as described above can be produced in large quantities and thus effectively used for medical or cosmetic purposes.

[0052] Continuous concentration system for producing ultra-high concentration medical exosome solutions

[0053] Another aspect of the present disclosure is to provide a continuous concentration system for producing ultra-high concentrated medical exosome solutions.

[0054] Conventional techniques for concentrating exosomes currently include ultracentrifugation, ultrafiltration, chromatography, and precipitation methods on cell-secreted exosomes from culture solutions obtained during cell culture.

[0055] However, compared with the initial capacity, the conventional technologies reported so far for exosome production can only achieve a concentration of 25 to 100 times higher and can only produce a maximum of 2.0 × 10 10 The exosomes have a concentration of 1.0×10 particles / mL. They are suitable for applications that have effective efficacy even at low concentrations (such as cosmetics), but for clinical applications at low concentrations or low volumes (such as local injection into the joint cavity in a very small volume), additional concentration procedures may be required. For a specific example, when 1.0×10 12 Currently, if a single exosome is an effective therapeutic dose, 50 mL would have to be administered to be effective, and methods capable of administering such large volumes are only possible through intravenous injection. In addition, low-concentration exosome therapeutic agents are very likely to stray from the site of application during local injection, which can present a disadvantage in reducing the therapeutic efficacy of the exosomes.

[0056] Currently, producing a high-concentration exosome solution requires an additional concentration process, which increases the number of steps and processing time. These steps significantly impact production manpower and costs, and increasing the number of steps leads to decreased productivity. Furthermore, factors such as sample contamination due to external factors, such as the movement of intermediate products or increased process exposure, can reduce the stability of the therapeutic agent.

[0057] The present disclosure provides a method for producing an ultra-high concentration exosome solution in a continuous process by injecting a solution containing exosomes (eg, a stem cell culture solution) as a raw material substance into a continuous concentration system, which solves such conventional problems.

[0058] Specifically, the present disclosure provides a method for producing an ultra-high concentration exosome solution, which includes injecting a raw material solution containing exosomes into a multiple TFF continuous concentration device, in which n TFF devices are connected as a system isolated from the outside; and continuously performing n concentration processes (here, n is an integer from 1 to 10) using the multiple TFF continuous concentration devices in a closed system.

[0059] "Continuous concentration system" means that multiple concentration processes are carried out continuously in a closed system by omitting the intermediate product recovery and raw material injection processes in two or more TFF concentration processes, such as Figures 12 to 14 shown.

[0060] Specifically, the dual and multiple TFF continuous concentration systems are constructed as a system isolated from the outside, and without recovering the product after the primary concentration, the initially injected raw material culture solution is simultaneously subjected to a secondary or multi-stage concentration process to produce the final raw material.

[0061] This system minimizes contamination caused by external factors such as migration of intermediate products, thereby showing improvements in the stability and quality of the efflux solution.

[0062] Even in conventional exosome extraction technologies, there are examples of adding a second stage of TFF concentration to extract high-concentration exosomes. However, in the present disclosure, dual or multiple concentration processes are performed simultaneously, thereby reducing process time and increasing the concentration of the final product by 25 times or more. Therefore, it can be considered different from simply adding a conventional concentration process.

[0063] Furthermore, the present disclosure allows for system scale-up, and the culture solution is completely protected from external exposure during the concentration process, ensuring pharmaceutical stability, making it suitable for pharmaceutical GMP applications. In particular, the present system does not require process operations in a BSC when applying GMP, a factor that reduces processability and operational difficulty. However, the present system allows for external BSC operations after obtaining APV certification.

[0064] The present disclosure consists of 1 TFF filter per concentrating section as well as 1 or more raw material injection vessels, 1 recovery vessel and a manifold.

[0065] The present disclosure performs double or multiple concentration sections consecutively.

[0066] 1) For the primary TFF filter unit, select the filter capacity based on scale

[0067] 2) The secondary TFF filter component has a scale of 1 / 10 to 1 / 50 times that of the primary filter component, and the filter capacity is selected according to the scale.

[0068] 3) The (n)th TFF filter component has a scale 1 / 10 to 1 / 50 times that of the (n-1)th filter component, and the filter capacity is selected according to the scale.

[0069] Depending on the purpose and scale, 2 to 5 filter members may be applied, but preferably, 2 to 3 members may be applied. However, it is not limited thereto.

[0070] A single filter member performs concentration 5 to 50 times based on the initial weight, but preferably, performs concentration 10 to 30 times. However, it is not limited thereto.

[0071] The TFF filter can be used in both sheet and hollow fiber types, but preferably performs concentration 10 to 30 times. However, it is not limited thereto.

[0072] The TFF filter can be selected based on capacity, but one should be selected with a shear rate of 1,000 to 6,000 s-1 during operation, and preferably, one should be selected with a shear rate of 3,000 to 5,000 s-1.

[0073] In the present disclosure, the concentration of exosomes finally obtained during the (n)th consecutive concentration theoretically provides a concentration of 25 to 50n times, or preferably, a concentration of 25 to 2500 times, or more preferably, a concentration of 50 to 100 times, based on the initial raw material weight.

[0074] Specifically, the final exosome product obtained in the present disclosure is 10 7 to 10 13 The exosome concentration of particles / mL is preferably 2.0×10 10 to 5.0×10 10 The exosome concentration of particles / mL was generated. However, in the present disclosure, it is not limited thereto.

[0075] On the other hand, the protein concentration of the final exosome product obtained in the present disclosure may be 1 to 1000 μg / mL, preferably 100 to 500 μg / mL, more preferably 300 to 400 μg / mL. However, in the present disclosure, it is not limited thereto.

[0076] As a specific example of the present disclosure, the system provides an injectable formulation containing a high concentration of stem cell exosomes for local injection.

[0077] Injectable preparations may have a 1.0×10 7 to 1.0×10 13 The exosome concentration ranges from 0.1 to 100 mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Schematic diagram of stem cell exosomes.

[0079] Figure 2 is a schematic diagram of a functional exosome.

[0080] Figure 3 Schematic diagram of tangential flow filtration during the functional exosome reaction process.

[0081] Figure 4Schematic diagram of functional exosome filtration and tangential flow filtration during the concentration process and washing process.

[0082] Figure 5 Shown are the morphology and fluorescence expression test results of DiD fluorescently labeled functional exosomes.

[0083] Figure 6 is a graph showing the ratio of the number of particles having a size of 50 to 200 nm among particles finally obtained through the processes of tangential flow filtration, ultracentrifugation, and membrane filter filtration.

[0084] Figure 7 Shown are the results of recovering fluorescently labeled exosomes produced by tangential flow filtration, ultracentrifugation, and membrane filter filtration using 1.5 mL tubes.

[0085] Figure 8 Shown are the results of determining the intracellular penetration of fluorescently labeled exosomes using confocal fluorescence microscopy.

[0086] Figure 9 Shown are the results of determining the changes in the physical properties of PEGylated exosomes produced by the tangential flow filtration process by zeta potential.

[0087] Figure 10 Shown are the results of determining changes in the physical properties of PEGylated exosomes produced by tangential flow filtration by DLS.

[0088] Figure 11 The UV-visible spectrophotometer is used to determine the dispersion of 10 8 to 10 10 Figure 3 Drug loading inside exosomes of 1 mL of DPBS per exosome.

[0089] Figure 12 is a schematic diagram showing the process for producing functional exosomes from cell culture solution using a continuous TFF process.

[0090] Figure 13 is a schematic diagram showing the process for producing functional exosomes from cell culture solution using a continuous TFF process.

[0091] Figure 14 is a schematic diagram showing the process for producing high concentrations of functional exosomes using a continuous TFF process.

[0092] Figure 15 Shown are the results of determining the yield of exosomes obtained by the continuous TFF process as the amount of exosomes and protein produced compared to the initial CM amount. DETAILED DESCRIPTION

[0093] Hereinafter, the present disclosure will be described in more detail by the following examples. However, the following examples are merely illustrative of the content of the present disclosure and do not limit the scope of the present disclosure. Contents that can be easily inferred by those skilled in the art from the detailed description and examples of the present disclosure are understood to belong to the scope of the present disclosure.

[0094] Example

[0095] 1. Culture of Human Adipose-Derived Stem Cells

[0096] To extract exosomes from human adipose-derived stem cells, human adipose-derived stem cells subcultured to passages 3 to 7 were cultured for 6 hours to 3 days, and the cell culture solution was recovered. The recovered cell culture supernatant was filtered through a 0.2 μm filter to remove cell debris and impurities.

[0097] 2. Extraction of Exosomes from Human Adipose-derived Stem Cells

[0098] Exosomes were extracted and purified from the human adipose-derived stem cell culture solution obtained in 1 using a tangential flow filtration system. A filter with a filtration capacity of 100 or 500 kDa was used as a filter for the tangential flow filtration system, and the cell culture solution was concentrated 10-fold or 100-fold to recover the exosomes. To improve the purity of the exosomes, the recovered exosomes were diluted 10-fold to 100-fold in DPBS and concentrated again using a filter with a filtration capacity of 100 or 500 kDa. The extraction process was carried out in Figure 3 and 4 Shown in.

[0099] 3. Characterization of Human Adipose-Derived Stem Cell Exosomes

[0100] The physical and biochemical properties of exosomes extracted from human adipose-derived stem cells by 2 were determined by protein quantification using nanoparticle tracking analysis (NTA) and bicinchonic acid (BCA) assays. The size of the exosome particles extracted in 2 was determined to be 50 to 200 nm.

[0101] 4. Generation of Fluorescently Labeled Functional Exosomes

[0102] (1) Using Tangential Flow Filtration to Produce Fluorescently Labeled Functional Exosomes (Example)

[0103] TFF device for generating fluorescently labeled functional exosomes in Figure 3 and Figure 4 1mL of the solution has a concentration of 5.0×10 9The exosomes were transferred to a 1.5 mL tube with 100 μL of fluorescent labeling material (Vybrant™ DiD cell labeling solution, ThermoFisher Scientific, V22887). The mixture was diluted 10-fold to 100-fold in DPBS and poured into the reservoirs of the TFF device and mixed.

[0104] use Figure 3 The TFF apparatus shown in the figure is used to circulate and react the exosomes and fluorescent marker material. The tube connected to the P (permeation) direction of the relative TFF filter and the tube connected to the reservoir are blocked by means of a clamp to prevent the reactants from escaping to the outside or mixing with the external buffer solution. The reservoir contains PBS for washing. Pump 1 is operated at a speed of 10 to 100 cc / minute for 10 seconds to 1 hour, so that the mixed solution of the exosomes and fluorescent marker material in the reservoir circulates inside the TFF filter. In this process, the fluorescent marker material is loaded on the exosome membrane, and the exosome surface is fluorescently labeled.

[0105] After the above process, a washing process is performed to remove the remaining fluorescent labeling material. Figure 4 As shown in , all clamps are removed. Pump 1 runs for 1 minute to 10 minutes at a speed of 10 to 250cc / minute, and pump 2 runs for 1 minute to 10 minutes at a speed of 5 to 10 to 50cc / minute. Pump 1 is used to circulate the mixed solution in TFF and the reservoir, and pump 2 is used to deliver the PBS used for washing to the reservoir, and to collect the waste solution containing the remaining fluorescent marker material. During this process, the exosomes larger than the TFF filter membrane pores are not filtered by the filter, but the fluorescent marker material smaller than the membrane pores is filtered, and therefore, the unreacted fluorescent marker material that does not react with the exosomes is removed, and only fluorescently labeled exosomes can be obtained. In order to determine the scavenging effect, samples are obtained according to 1 minute 1mL, and absorbance is measured at 450nm using a UV-visible spectrophotometer. The exosomes and waste and PBS finally separated are collected per unit volume, and the absorbance at 450nm is measured in the same manner ( Figure 5 ).

[0106] (2) Using ultracentrifugation to generate fluorescently labeled functional exosomes (Comparative Example 1)

[0107] A mixture of exosomes and fluorescent labeling materials was produced by the same method as (1). Specifically, 1 mL of a 5.0×10 9The exosomes were transferred to a 1.5 mL tube with 100 μL of fluorescent labeling material (Vybrant™ DiD cell labeling solution, ThermoFisher Scientific, V22887). The mixture was diluted 10-fold to 100-fold in DPBS and poured into the reservoirs of the TFF device and mixed.

[0108] Then, the mixture was centrifuged at 80,000×g to 150,000×g to spin down the fluorescently labeled exosomes. The fluorescently labeled exosomes were dispersed in 1 mL of DPBS to obtain a fluorescently labeled exosome solution ( Figure 6 , 7).

[0109] (3) Producing Fluorescently Labeled Functional Exosomes Using a Membrane Filter Method (Comparative Example 2)

[0110] A mixture of exosomes and fluorescent labeling materials was produced by the same method as (1). Specifically, 1 mL of a 5.0×10 9 The exosomes were transferred to a 1.5 mL tube with 100 μL of fluorescent labeling material (Vybrant™ DiD cell labeling solution, ThermoFisher Scientific, V22887). The mixture was diluted 10-fold to 100-fold in DPBS and poured into the reservoirs of the TFF device and mixed.

[0111] Then, a membrane filter with a filtration capacity of 3 to 50 kDa ( The mixture was centrifuged at a rate of 1,000 × g to 4,000 × g for 30 minutes using Ultra-15 Centrifugal Filter Units, Merck, and the fluorescently labeled exosomes were concentrated and purified. The fluorescently labeled exosomes were recovered using a 1.5 mL tube to obtain a fluorescently labeled exosome solution ( Figure 6 , 7).

[0112] 5. Characterization of Fluorescently Labeled Functional Exosomes

[0113] In order to determine the fluorescent labeling ability of the fluorescently labeled functional exosomes produced in 4(1), 10 8 to 10 10 1 mL of DPBS containing 1 exosome was mixed with 9 mL of cell culture solution and treated on human keratinocytes (HaCaT) for 1 hour. For human keratinocytes treated with exosomes, intracellular penetration of fluorescently labeled exosomes was determined using confocal fluorescence microscopy ( Figure 8 ).

[0114] 6. Using Tangential Flow Filtration to Generate PEGylated Functional Exosomes

[0115] PEGylated exosomes were produced by a method similar to that of 4(a). 1 mL of 5.0×10 9 The exosomes at 100 μL of 1 to 1,000 mg / mL of particles / mL and 10 μL of 1 to 1,000 mg / mL polyethylene glycol (PEG) were transferred to a 1.5 mL tube. The mixture was diluted 10 to 100 times the volume in DPBS and poured into the reservoirs in the TFF apparatus and mixed. To induce chemical binding of the exosomes and PEG, a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) crosslinking process was performed. 500 μL of 1 to 100 mM EDC and 500 μL of the same concentration of NHS were added to the exosome / PEG mixture and allowed to react in the reservoir for approximately 1 to 30 minutes.

[0116] After the above process, a washing process is performed to remove the remaining fluorescent labeling material. Figure 4 As shown in , all clamps are removed. Pump 1 is operated at a speed of 10 to 250 cc / min for 30 minutes to 3 hours, and pumps 2 and 3 are operated at a speed of 5 to 10 to 50 cc / min for 30 minutes to 3 hours. Pump 1 is used to circulate the mixed solution between the TFF and the reservoir, while pumps 2 and 3 are used to deliver PBS for washing to the reservoir, and to collect the waste solution containing the remaining fluorescent labeling material, respectively. During this process, exosomes larger than the TFF filter membrane pores are not filtered by the filter, but chemicals smaller than the membrane pores are filtered, and therefore, unreacted materials that have not reacted with the exosomes are removed, and only fluorescently labeled exosomes can be obtained.

[0117] 7. Characterization of PEGylated Functional Exosomes

[0118] The changes in the physical properties of exosomes extracted from human adipose-derived stem cells were determined by DLS and zeta potential to determine whether the surface of the exosome particles was modified. It was determined that the PEGylated functional exosomes had a larger particle size distribution ( Figure 9 , 10).

[0119] 8. Using Tangential Flow Filtration to Generate Drug-Loaded Functional Exosomes

[0120] Drug-loaded exosomes were produced and purified by a method similar to that in 4(a). 9100 μL of 1 to 1,000 μg of doxorubicin was added to each exosome at a concentration of 100 particles / mL. The mixture was diluted 10- to 100-fold in DPBS and poured into the disposable bags in the TFF apparatus and mixed. For drug loading of the exosomes, the disposable bags containing the mixture were immersed in a sonicated water bath maintained at 4°C or below.

[0121] use Figure 3 The TFF device shown in the embodiment of the present invention is a process for reacting by circulating a mixture of exosomes and drugs. Then, the disposable bag is kept in a water bath that is slightly sonicated. The tube connected in the P (permeation) direction relative to the TFF filter and the tube connected to the reservoir are blocked by means of a clamp to prevent the reactants from escaping to the outside or mixing with external buffer, and the reservoir has PBS for washing. Pump 1 is operated at a speed of 10 to 100cc / minute for 10 seconds to 1 hour, so that the mixed solution of the exosomes in the reservoir and the drug circulates inside the TFF filter. In this process, the drug is loaded in the exosomes.

[0122] After the above process, a washing process is performed to remove the remaining drugs. Figure 4 As shown in , all clamps are removed. Pump 1 runs for 30 minutes to 3 hours at a speed of 10 to 250cc / minute, and pumps 2 and 3 run for 30 minutes to 3 hours at a speed of 5 to 10 to 50cc / minute. Pump 1 is used for the purposes of circulating the mixed solution in TFF and the reservoir, while pump 2 and pump 3 are used for the purposes of delivering the PBS for washing to the reservoir, and for the purposes of collecting the waste solution containing remaining medicine. During this process, the exosomes larger than the TFF filter membrane pores are not filtered by the filter, while the medicine smaller than the membrane pores is filtered, and therefore, the medicine not loaded on the exosomes is removed, and the exosomes only loaded with medicine can be obtained.

[0123] 9. Characterization of drug-loaded functional exosomes

[0124] To determine the drug loading of drug-loaded exosomes, a UV-visible spectrophotometer was used using a 8 to 10 10 The drug loading inside the exosomes was determined by 1 mL DPBS per exosome ( Figure 11 ).according to Figure 11 , it was determined that the exosomes were 5×10 9 Each particle was loaded with about 20 μg of doxorubicin.

[0125] 10. Using a continuous TFF process to extract high-concentration human adipose-derived stem cell exosomes

[0126] Exosomes were extracted and purified from the human adipose-derived stem cell culture solution obtained in 1 using a continuous tangential flow filtration system. As a filter for the primary tangential flow filtration method, a filter with a filtration capacity of 100 or 500 kDa was used, and the cell culture solution was concentrated 10-fold or 100-fold to recover the exosomes. To improve the purity of the exosomes, the recovered exosomes were diluted 10-fold to 100-fold in DPBS and concentrated using a filter with a filtration capacity of 100 or 500 kDa. The extraction process was carried out in Figure 12 、 13 The yield of exosomes obtained by the continuous TFF process was determined by the amount of exosomes and protein produced compared to the initial CM amount ( Figure 15 ).

Claims

1. A method for preparing functional exosomes by modifying exosomes with functional materials using a tangential flow filtration (TFF) device, The method comprises the following steps: a reaction step of circulating and mixing the exosome with the functional material within the TFF device; filtration and concentration steps to remove unreacted functional materials; as well as a washing step of replacing the solvent in the mixed solution, wherein the reaction step is performed by circulating the exosome and the functional material between the reservoir and the TFF filter in the TFF apparatus via a tube connected in a permeation direction relative to the TFF filter and a tube connected to a reservoir by blocking with a clamp, wherein the filtration and concentration steps, and the washing step are performed simultaneously by removing all clamps to open the tube connected to the permeation direction of the TFF filter and the tube connected to the reservoir, wherein the reacting step, the filtering and concentrating step, and the washing step are performed using the same TFF apparatus, the same TFF apparatus being a first TFF apparatus, and The method does not use a separate batch reactor to react the exosome with the functional material. 2 . The method according to claim 1 , wherein the functional material is one or more selected from the group consisting of: a biocompatible polymer, a protein drug, a chemical drug, and a labeling molecule. 3 . The method according to claim 1 , wherein the reacting step is performed while the exosome and the functional material are circulated by a pump of the TFF device.

4. The method according to claim 1, wherein the method does not use a separate stirring device and a separate washing device other than the TFF apparatus. The method according to claim 1 , wherein the method continuously performs the reacting step, the filtering and concentrating step, and the washing step using the TFF device. The method of claim 1 , wherein the method is used to produce the functional exosomes on a large scale.

7. The method according to claim 1, further comprising the step of injecting the concentrated functional exosomes obtained from the first TFF device into a multiple TFF continuous concentration device to further concentrate the exosomes. The multiple TFF continuous concentration device comprises n TFF devices, and the n TFF devices are connected to form a closed system isolated from the outside, and n concentration processes are continuously performed in the closed system, and n is an integer from 1 to 10.

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