WO2021221368A1 - Method for filtration of cell-derived vesicle - Google Patents

Method for filtration of cell-derived vesicle Download PDF

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WO2021221368A1
WO2021221368A1 PCT/KR2021/004883 KR2021004883W WO2021221368A1 WO 2021221368 A1 WO2021221368 A1 WO 2021221368A1 KR 2021004883 W KR2021004883 W KR 2021004883W WO 2021221368 A1 WO2021221368 A1 WO 2021221368A1
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cell
derived
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cells
derived vesicles
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French (fr)
Korean (ko)
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박진희
회이총라우
오승욱
배신규
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주식회사 엠디뮨
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/02—Hollow fibre modules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A20/00—Water conservation; Efficient water supply; Efficient water use
    • Y02A20/124—Water desalination
    • Y02A20/131—Reverse-osmosis

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  • the present invention relates to a method for isolating and selectively purifying cell-derived vesicles with high purity from a suspension containing cell-derived vesicles, and more particularly, to cell-derived vesicles from a suspension containing cell-derived vesicles. It relates to a method for efficiently fractionating, separating and selectively purifying impurities such as debris, waste products, proteins and large particles.
  • cell secretome contains various bioactive factors that control cell behavior. Because it contains 'extracellular vesicle', research on its components and functions is being actively conducted.
  • extracellular vesicles Cells release various membrane types of ERs to the extracellular environment, and these ERs are commonly referred to as extracellular vesicles.
  • the extracellular vesicles are also called cell membrane-derived ERs, ectosomes, shedding vesicles, microparticles, exosomes, and the like, and in some cases, they are used separately from exosomes.
  • Exosomes are endoplasmic reticulum with a size of several tens to hundreds of nanometers composed of a double phospholipid membrane identical to the structure of the cell membrane, and contain proteins, mRNA, miRNA, etc. called exosome cargo inside.
  • Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differently regulated according to the environment of secretory cells.
  • Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells.
  • Exosomes contain specific genetic material and bioactive factors according to the nature and state of the cell from which they are derived. In the case of proliferating stem cell-derived exosomes, they control cell behaviors such as cell migration, proliferation and differentiation, and reflect the characteristics of stem cells related to tissue regeneration.
  • a cell-derived vesicle manufactured by extruding a nucleated cell unlike the existing naturally secreted microvesicle, has a characteristic of maintaining topology such as a cell membrane. confirmed that there is.
  • Ultracentrifugation is the most widely used method so far to isolate exosomes, extracellular vesicles, or cell-derived vesicles. There are disadvantages. In addition, the ultracentrifugation method has a disadvantage in that it may damage exosomes, extracellular vesicles, or cell-derived vesicles during the separation process, which may interfere with subsequent analysis processes or applications.
  • Ultrafiltration can be used in conjunction with ultracentrifugation to increase the purity of exosomes, extracellular vesicles, or cell-derived vesicles, but the exosomes, extracellular vesicles or cell-derived vesicles adhere to the filter and separate There is a problem in that the subsequent yield is low.
  • the immunoaffinity separation method has the advantage of high specificity by attaching the antibody to exosomes, extracellular vesicles, or cell-derived vesicles. This has the disadvantage of being expensive, and it is an unsuitable method for scale-up.
  • exosome separation kits such as exosome precipitation, total exosome isolation kit, or polymer based precipitation are commercially available. Although it is sold, it is easy to use, but the reagent is expensive, so it can be used to isolate exosomes or extracellular vesicles at the laboratory level, but there is a problem that is not suitable for isolating and purifying exosomes or extracellular vesicles in large quantities.
  • an object of the present invention is to provide a method for efficiently separating and purifying cell-derived vesicles from a sample containing cell-derived vesicles.
  • the present invention maintains a) a sample containing a cell-derived vesicle at a flow rate of 60 to 120 ml/min and a transmembrane pressure of 0.1 to 0.3 bar, molecular weight Using a tangential flow filtration (TFF) filter having a molecular weight cutoff (MWCO) of 500 to 1,000 kDa, performing cross flow filtration; and b) filtering using a filter having a pore size of 0.2 to 0.5 ⁇ m, wherein the cross-flow filtration in step a) is 1) through ultrafiltration to 20 to 40% of the initial volume of the sample.
  • TMF tangential flow filtration
  • MWCO molecular weight cutoff
  • the present invention provides a cell-derived vesicle isolated and purified by the above method.
  • cell-derived vesicle separation and purification method Using the cell-derived vesicle separation and purification method according to the present invention, cell-derived vesicles can be isolated and purified with high yield and high purity.
  • 1 is a diagram schematically showing the separation and purification process of mesenchymal stem cell-derived vesicles.
  • FIG. 2 is a diagram showing the ratio of the number of particles, protein, and DNA compared to the cell-derived vesicle (Crude CDV) before purification at each purification step by varying the transmembrane pressure during cross flow filtration to 0.1, 0.2, and 0.5 bar. .
  • 3 is a flow rate of 60, 80, 100, and 120 mL/min during cross-flow filtration, and the ratio of the number of particles, protein, and DNA compared to the cell-derived vesicle (Crude CDV) before purification for each purification step is compared. is the diagram shown.
  • FIG. 4 is a diagram showing the ratio of the number of particles, protein, and DNA compared to the cell-derived vesicle (Crude CDV) prior to purification by varying the MWCO of the TFF filter to 750 kDa and 100 kDa during cross flow filtration, and for each purification step. .
  • 5 is a diagram comparing the yield (particle/cell) and purity (particle/ug) by varying the dilution factor of the ultrafiltration process by 14 times, 20 times, 22 times, and 23 times.
  • FIG. 6 is a diagram comparing the ratio of the number of particles, protein, and DNA to cell-derived vesicles (Crude CDV) prior to purification in each purification step by varying the volume of the buffer solution used in the diafiltration process by 1 to 6 times; am.
  • FIG. 7 is a diagram comparing the number of cell-derived vesicle particles, the number of proteins, the size, and the polydispersity index (PDI) before and after each filtration by varying the size of the pores of the filtration filter to 0.45um and 0.22um.
  • PDI polydispersity index
  • Example 8 is a diagram showing the yield when the mesenchymal stem cell-derived vesicles are isolated and purified by the method of Example 2.
  • Example 9 is a diagram showing the purity when the mesenchymal stem cell-derived vesicles are isolated and purified by the method of Example 2.
  • FIG. 10 is a diagram showing the results of comparing the yield and purity of cell-derived vesicles according to various purification methods gUC and UC and the purification method (TFF) of the present invention.
  • the present invention a) maintains a flow rate of 60 to 120 ml/min and a transmembrane pressure of 0.1 to 0.3 bar for a sample containing a cell-derived vesicle, and molecular weight cutoff (MWCO) ) using a 500 to 1,000 kDa cross-flow filtration (tangential flow filtration, TFF) filter, performing cross-flow filtration; and b) filtering using a filter having a pore size of 0.2 to 0.5 ⁇ m, wherein the cross-flow filtration in step a) is 1) through ultrafiltration to 20 to 40% of the initial volume of the sample.
  • MWCO molecular weight cutoff
  • the cell-derived vesicle refers to a vesicle artificially manufactured in a nucleated cell, is separated from the cell membrane in almost all types of cells, and has a double phospholipid membrane, which is the structure of the cell membrane.
  • the cell-derived vesicle of the present invention may have a micrometer size, for example, 0.03 to 1 ⁇ m.
  • the cell-derived vesicle of the present invention is distinguished from the naturally secreted vesicle, and may be prepared by extruding a sample containing cells into micropores, and preferably, the micropore size is larger than the micropore size. It may be manufactured by sequential extrusion with a small size, preferably a membrane filter having a micropore size of 9 to 11 ⁇ m, 2 to 4 ⁇ m, and 0.6 to 0.2 ⁇ m, more preferably 10 ⁇ m, It may be manufactured by sequentially extruding a membrane filter of 3 ⁇ m and 0.4 ⁇ m.
  • the cells for preparing the cell-derived vesicles may include without limitation as long as they are nucleated cells, but preferably stem cells, acinar cells, myoepithelial cells, red blood cells, monocytes, dendritic cells, and natural killer cells. And it may be any one or more selected from the group consisting of platelets.
  • the stem cells may be any one or more selected from the group consisting of mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells and salivary gland stem cells, more preferably mesenchymal stem cells, even more preferably the mesenchymal stem cells It may be adipose, bone marrow, umbilical cord or umbilical cord blood-derived mesenchymal stem cells.
  • the 'vesicle' of the present invention is separated from the inside and outside by a lipid double membrane composed of the cell membrane component of the derived cell, and has cell membrane lipids, cell membrane proteins, nucleic acids and cell components of the cell, and is larger in size than the original cell. means small, but not limited thereto.
  • the desalting and buffer exchange may be carried out continuously or non-continuously, but preferably may be carried out continuously.
  • tangential flow filtration refers to a filtration method in which the direction in which the sample is filtered and the direction in which the sample is supplied are perpendicular.
  • the flow rate means the volume of fluid passing per unit time, preferably the flow rate may be 60 to 120 ml / min, preferably 70 to 90 ml / min, more preferably Preferably, it may be 80 ml/min in consideration of the property that the surface protein of the cell-derived vesicle is vulnerable to shear stress.
  • the transmembrane pressure means a pressure difference with the filtration filter as a boundary, and preferably, the transmembrane pressure may be 0.1 to 0.3 bar, more preferably 0.2 bar. When the transmembrane pressure is 0.2 bar, proteins and DNA present in the sample can be most effectively removed.
  • the molecular weight cutoff means the lowest molecular weight of the solute in which 90% of the solute is maintained by the filtration filter, preferably the molecular weight cutoff may be 500 to 1,000 kDa, preferably Preferably, it may be 700 to 800 kDa, and more preferably 750 kDa. When the molecular weight cutoff is 750 kDa, it is possible to separate and purify cell-derived vesicles with high purity.
  • the ultrafiltration refers to a filtration method in which extremely fine particles are separated by filtration through a filtration membrane having very small pores, and the solution that does not pass through the filtration membrane is concentrated.
  • the ultrafiltration may be a primary concentration of 20 to 40% of the initial volume, and a secondary concentration of 3 to 7% of the initial volume after desalting and buffer exchange, most preferably
  • the primary concentration may be 25% of the initial volume
  • the secondary re-concentration may be preferably 4 to 6%, most preferably 5% of the initial volume after desalting and buffer exchange.
  • the desalting and buffer exchange may be performed by diafiltration using a buffer solution, and in the present invention, the desalting and buffer exchange are 5 based on the volume of the concentrated sample. It may be diafiltration using a buffer solution having a volume of to 7 times, and most preferably, diafiltration using a buffer solution having a volume of 6 times the volume of the concentrated sample. have.
  • step 2) of the present invention is a step of performing desalting and buffer exchange using a buffer solution having a volume 5 to 7 times the volume based on the finished volume, and step 3) is 3 to 7 of the initial volume of the sample.
  • % may be a step of re-concentration through ultrafiltration.
  • primary ultrafiltration is performed to concentrate to 25% of the initial volume, and desalting and buffer exchange are performed through diafiltration using a buffer having a volume of 6 times the volume of the sample after concentration is completed.
  • desalting and buffer exchange are performed through diafiltration using a buffer having a volume of 6 times the volume of the sample after concentration is completed.
  • more than 80% of protein is removed, and 95% of DNA is removed during nuclease treatment as described below. can be achieved.
  • the method for separating and purifying the cell-derived vesicle further comprises treating a nuclease, Tris-HCl and magnesium chloride before performing the cross flow filtration of step a).
  • the method may further comprise treating a sample containing a cell-derived vesicle with a nuclease, 40 to 60 mM Tris-HCl, and 1 to 3 mM magnesium chloride in proportion to the amount of DNA in the sample. have.
  • the filter of step b) may have a pore size of 0.3 to 0.5 ⁇ m, preferably 0.45 ⁇ m.
  • the size of the pores is 0.3 to 0.5 ⁇ m, particularly 0.45 ⁇ m, the particle loss rate of the cell-derived vesicle is remarkably low, and the size and uniformity of the cell-derived vesicle may be maintained.
  • the cell-derived vesicles separated and purified by the cell-derived vesicle separation and purification method of the present invention have a size of 100 to 200 nm, and a polydispersity index (PDI) of 0.2 to 0.5, preferably For example, it may be 0.2 to 0.3.
  • PDI polydispersity index
  • the method for isolating and purifying cell-derived vesicles of the present invention most preferably comprises: a) adding nuclease, 50 mM Tris-HCl and 2 mM magnesium chloride to a sample containing cell-derived vesicles in proportion to the amount of DNA in the sample to process; b) centrifugation at 3000 x G conditions for 10 minutes at a room temperature of 20 to 25 °C; c) cross flow filtration of a sample containing cell-derived vesicles with a flow rate of 80 ml/min and a transmembrane pressure of 0.2 bar, and a molecular weight cutoff (MWCO) of 750 kDa (tangential flow filtration, TFF) using a filter, performing cross-flow filtration; d) centrifugation at 3000 x G conditions for 10 minutes at room temperature of 20 to 25 °C; and e) filtering using a filter having a pore size of 0.45 ⁇ m, wherein the
  • the present invention provides a cell-derived vesicle isolated and purified by the above method.
  • the cell-derived vesicle may have a size of 100 to 200 nm, and a polydispersity index (PDI) of 0.2 to 0.5, preferably 0.2 to 0.3.
  • PDI polydispersity index
  • Example 1 Preparation of mesenchymal stem cell-derived vesicle suspension before purification
  • a vesicle suspension was prepared from umbilical cord blood mesenchymal stem cells (UC MSC) by extrusion.
  • Mesenchymal stem cells cultured in stem cell complete growth medium are washed with phosphate buffered saline (PBS), and the washed stem cells are resuspended in PBS at a concentration of 0.25 to 1 x 10 6 cells/ml ( resuspension).
  • the suspension solution was passed through a membrane filter having a micropore size of 10 ⁇ m using a cell extruder, and then passed through a membrane filter having a micropore size of 3 ⁇ m, followed by a micropore size of 0.4 ⁇ m.
  • a suspension of mesenchymal stem cell-derived vesicles (Crude UCMSC-CDV) was prepared.
  • Example 2 Isolation and purification method of mesenchymal stem cell-derived vesicles
  • mesenchymal stem cell-derived vesicles (Crude UCMSC-CDV) produced through a cell extruder, 100-200 nm using Tangential Flow Filtration (TFF) Mesenchymal stem cell-derived vesicles were purified to have a size of and PDI to have a uniformity of 0.2 to 0.3. Specifically, mesenchymal stem cell-derived vesicles were isolated and purified using Spectrum® KR2i TFF Systems (Repligen), a hollow fiber filter (Repligen, 750 kDa, 0.01 m 2 ).
  • benzonase nuclease (Merck, 71206-3), 50 mM Tris-HCl (Teknova, 50-843-335), 2mM magnesium chloride (Magnesium chloride, SIGMA-ALDRICH, M1028-100mL) was treated for 90 minutes, 37 °C condition.
  • the aggregates in the vesicle suspension were removed by centrifugation at 3000 x G conditions for 10 minutes at room temperature.
  • the suspension was concentrated at a flow rate of 80 mL/min while maintaining a transmembrane pressure of 0.2 bar, and impurities were filtered off.
  • the suspension was concentrated until the volume was about 1/4 of the volume, and then impurities were removed through desalting and buffer exchange in the diafiltration process. Desalting and buffer exchange were performed continuously, and a buffer solution (PBS) having at least 6 times the volume of the concentrated volume was used.
  • PBS buffer solution
  • the separated/purified mesenchymal stem cell-derived vesicles were centrifuged at room temperature for 10 minutes at 3000 x G to remove aggregates, filtered through a 0.45um syringe filter (pall, 4614), and finally mesenchymal stem cell-derived vesicles were removed. obtained. Thereafter, the physical properties of the mesenchymal stem cell-derived vesicles purified through DLS, NTA, and measurement were analyzed, and the removal rate of impurities compared to before purification was confirmed through quantification of protein, DNA, and benzonase.
  • the process of separation and purification of the mesenchymal stem cell-derived vesicles as described above is schematically shown in FIG. 1 .
  • Example 3 Selection of conditions for isolation and purification of mesenchymal stem cell-derived vesicles
  • the conditions of high flow rate and transmembrane pressure with high DNA and non-specific protein removal efficiency were selected.
  • the transmembrane pressure in the method of Example 2, the transmembrane pressure was changed to 0.1, 0.2, and 0.5 bar, and the ratio of the number of particles, protein, and DNA to cell-derived vesicle (Crude CDV) before purification for each purification step was calculated. Measurements were made as follows.
  • the number of particles compared to cell-derived vesicles was measured by NTA (Nanoparticle Tracking Analysis; Nanosight NS300, Malvern Panalytical, Westborough, MA, USA).
  • Protein concentration was measured using a QubitTM 4 fluorometer (Thermo Scientific, Waltham, MA, USA) and a QubitTM Protein Assay Kit (Thermo Fisher scientific). Samples and standard solutions were prepared by mixing 190 uL of working solution with 10 uL of sample and standard solution, respectively. Each mixture was vortexed for 2-3 seconds and incubated for 15 minutes at room temperature. After that, the standard solution and the sample were sequentially put into the equipment, and the protein concentration in the sample was measured.
  • the proportion of DNA was measured using a QubitTM 4 fluorometer (Thermo Scientific, Waltham, MA, USA) and a QubitTM HS dsDNA assay kit (Thermo Fisher scientific). Samples and standard solutions were prepared by mixing 190 uL of working solution with 10 uL of sample and standard solution, respectively. Each mixture was vortexed for 2-3 seconds and then incubated for 2 minutes at room temperature. Then, the standard solution and the sample were sequentially put into the equipment and the DNA concentration of the sample was measured.
  • transmembrane pressure which is the pressure applied to the membrane
  • the transmembrane pressure was 0.1 and 0.2 bar
  • the particle recovery rate was similar, but the protein and DNA removal rate was higher at 0.2 bar.
  • 0.2 bar corresponds to the best transmembrane pressure.
  • Example 3.1 As in Example 3.1, the flow rate was changed to 60, 80, 100, and 120 mL/min, and the ratio of the number of particles, protein, and DNA to the cell-derived vesicle (Crude CDV) before purification for each purification step was compared, and the The results are shown in FIG. 3 .
  • a flow rate of 80 mL/min was selected in consideration of the vulnerability of the surface protein of the cell-derived vesicle to shear stress.
  • the volume of the buffer solution used in the diafiltration process showing excellent desalting and buffer exchange effects in the cell-derived vesicle purification and separation method was selected. Desalting and buffer exchange are performed by varying the volume of the buffer solution by 1 to 6 times the volume of the concentrated sample, and the ratio of the number of particles, protein, and DNA to cell-derived vesicles (Crude CDV) before purification for each purification step. were compared, and the results are shown in FIG. 6 .
  • the size of the pore size of the filtration filter showing an excellent purification effect in cell-derived vesicle purification and separation was selected.
  • the size of the filtration filter pore size was 0.45um and 0.22um, and the number of cell-derived vesicle particles, the number of proteins, the size, and PDI (polydispersity index) were compared before and after each filtration. , the results are shown in FIG. 7 .
  • Example 4 Evaluation of yield and purity according to the method for isolation and purification of mesenchymal stem cell-derived vesicles
  • Example 2 In order to evaluate the purity of the mesenchymal stem cell-derived vesicles isolated and purified by the method of Example 2, the amounts of the cell-derived vesicles and the protein were measured in the same manner as in Example 3.1, and the purity was calculated. was graphed and shown in FIG. 9 .
  • cell-derived vesicles were obtained by the method of Example 2, it was confirmed that cell-derived vesicles could be obtained with a high purity of 3.86 X 10 9 ⁇ 7.37 X 10 8 particle/ug. .
  • the purity by the method of purifying the existing exosomes, exosome analogues or extracellular vesicles is shown in Table 2.
  • Example 2 of the present invention Using the purification method of Example 2 of the present invention, it was confirmed through a specific comparative experiment whether the cell-derived vesicles could be purified with better yield and higher purity than other purification methods.
  • conventional known exosome separation methods such as gradient ultracentrifugation (gUC) and ultracentrifugation (UC) were performed, and Example 2 of the present invention and particle yield and purity were compared.
  • the gUC method was performed by stacking Crude CDV on 50 and 10% Opti-prep solutions and ultracentrifugation at 120,000 g, 4° C. for 2 hr.
  • the UC method was performed by centrifuging Crude CDV at 3,000 g, 4° C. for 10 min, and then ultracentrifuging the supernatant sequentially at 10,000 g, 30 min and 120,000 g at 4° C., and 4° C. for 2 hr.
  • the results of comparison of particle yield and purity of the obtained stem cell-derived vesicles are shown in FIG. 10 .
  • the particle yield increased by about 1.5 to 2 times compared to other existing exosome separation methods, and the purity was about 1.3 to 3 times. It was confirmed that the fold increased.

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Abstract

The present invention relates to a method for isolating cell-derived vesicles at high purity and selectively filtering cell-derived vesicles from a suspension containing cell-derived vesicles and, more specifically, to a method for isolation and selective filtration of cell-derived vesicles from a suspension containing cell-derived vesicles through efficient discrimination against impurities cell debris, waste products, proteins, and macro-particles. Using the method for isolation and filtration of cell-derived vesicles according to the present invention, cell-derived vesicles can be isolated and filtered with high yield and high purity.

Description

세포 유래 베시클의 정제 방법Purification method for cell-derived vesicles
본 발명은 세포 유래 베시클이 포함된 현탁액으로부터 세포 유래 베시클을 고순도로 분리하고, 선택적으로 정제하는 방법에 관한 것으로서, 보다 상세하게는 세포 유래 베시클이 포함된 현탁액으로부터 세포 유래 베시클을 세포 잔해물, 노폐물, 단백질 및 거대 입자와 같은 불순물에 대해 효율적으로 분별하여 분리 및 선택적으로 정제하는 방법에 관한 것이다.The present invention relates to a method for isolating and selectively purifying cell-derived vesicles with high purity from a suspension containing cell-derived vesicles, and more particularly, to cell-derived vesicles from a suspension containing cell-derived vesicles. It relates to a method for efficiently fractionating, separating and selectively purifying impurities such as debris, waste products, proteins and large particles.
최근 세포 분비물(secretome)에 세포의 행동을 제어하는 다양한 생체활성인자가 포함되어 있다는 연구가 보고되고 있으며, 특히 세포 분비물 내에는 세포 간 신호전달 기능을 갖는 나노소포체인 '엑소좀(exosome)' 또는 '세포외소포체(extracellular vesicle)'가 포함되어 있어 그 성분과 기능에 대한 연구가 활발히 진행 중에 있다. Recently, research has been reported that cell secretome contains various bioactive factors that control cell behavior. Because it contains 'extracellular vesicle', research on its components and functions is being actively conducted.
세포는 세포외 환경에 다양한 막(membrane) 유형의 소포체를 방출하는데, 통상 이러한 방출 소포체들을 세포외 소포체(extracellular vesicle)라고 부르고 있다. 세포외 소포체는 세포막 유래 소포체, 엑토좀(ectosomes), 쉐딩 소포체(shedding vesicles), 마이크로파티클(microparticles), 엑소좀 등으로 불려지기도 하며, 경우에 따라서는 엑소좀과는 구별되어 사용되기도 한다.Cells release various membrane types of ERs to the extracellular environment, and these ERs are commonly referred to as extracellular vesicles. The extracellular vesicles are also called cell membrane-derived ERs, ectosomes, shedding vesicles, microparticles, exosomes, and the like, and in some cases, they are used separately from exosomes.
엑소좀은 세포막의 구조와 동일한 이중인지질막으로 이루어진 수십 내지 수백 나노미터 크기의 소포체로서 내부에는 엑소좀 카고(cargo)라고 불리는 단백질, mRNA, miRNA 등이 포함되어 있다. 엑소좀 카고에는 광범위한 신호전달 요소들(signaling factors)이 포함되며, 이들 신호전달 요소들은 세포 타입에 특이적이고 분비세포의 환경에 따라 상이하게 조절되는 것으로 알려져 있다. 엑소좀은 세포가 분비하는 세포 간 신호전달 매개체로서 이를 통해 전달된 다양한 세포 신호는 표적 세포의 활성화, 성장, 이동, 분화, 탈분화, 사멸(apoptosis), 괴사(necrosis)를 포함한 세포 행동을 조절한다고 알려져 있다. 엑소좀은 유래된 세포의 성질 및 상태에 따라 특이적인 유전물질과 생체활성 인자들이 포함되어 있다. 증식하는 줄기세포 유래 엑소좀의 경우 세포의 이동, 증식 및 분화와 같은 세포 행동을 조절하고, 조직 재생과 관련된 줄기세포의 특성이 반영되어 있다. Exosomes are endoplasmic reticulum with a size of several tens to hundreds of nanometers composed of a double phospholipid membrane identical to the structure of the cell membrane, and contain proteins, mRNA, miRNA, etc. called exosome cargo inside. Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differently regulated according to the environment of secretory cells. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. is known Exosomes contain specific genetic material and bioactive factors according to the nature and state of the cell from which they are derived. In the case of proliferating stem cell-derived exosomes, they control cell behaviors such as cell migration, proliferation and differentiation, and reflect the characteristics of stem cells related to tissue regeneration.
또한, 대한민국 등록특허(KR10-1314868)에서는 유핵세포를 압출하는 방법으로 제조한 세포 유래 베시클인 마이크로베시클이 기존에 존재하는 자연 분비 마이크로베시클과 달리, 세포막과 같은 토폴로지를 유지하는 특성이 있음을 확인하였다. In addition, in the Republic of Korea Patent (KR10-1314868), a cell-derived vesicle manufactured by extruding a nucleated cell, unlike the existing naturally secreted microvesicle, has a characteristic of maintaining topology such as a cell membrane. confirmed that there is.
이와 같은 엑소좀, 세포외소포체 또는 세포 유래 베시클을 분리하는 종래 기술로는 초원심분리법(ultracentrifugation), 밀도구배원심법(density gradient centrifugation), 초미세여과법(ultrafiltration), 사이즈 배제 크로마토그래피(size exclusion chromatography), 이온교환 크로마토그래피(ion exchange chromatography), 면역친화성 분리법(immunoaffinity capture), 미세유체기술 분리법(microfluidics-based isolation), 침전법(exosome precipitation), 총엑소좀 추출 키트(total exosome isolation kit), 또는 폴리머 기반 침전법(polymer based precipitation) 등이 있다. Conventional techniques for separating such exosomes, extracellular vesicles or cell-derived vesicles include ultracentrifugation, density gradient centrifugation, ultrafiltration, and size exclusion chromatography (size). exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit), or polymer-based precipitation.
초원심분리법(ultracentrifugation)은 엑소좀, 세포외소포체 또는 세포 유래 베시클을 분리하는데 지금까지 가장 널리 사용되었던 방법이나, 수율이 낮고, 분리하는데 시간이 많이 소요되며 노동집약적이고, 비싼 기기가 필요하다는 단점이 있다. 또한, 초원심분리법은 분리과정에서 엑소좀, 세포외소포체 또는 세포 유래 베시클에 손상을 줄 수 있어 후속 분석 과정이나 응용에 지장을 줄 수 있는 단점이 있다. Ultracentrifugation is the most widely used method so far to isolate exosomes, extracellular vesicles, or cell-derived vesicles. There are disadvantages. In addition, the ultracentrifugation method has a disadvantage in that it may damage exosomes, extracellular vesicles, or cell-derived vesicles during the separation process, which may interfere with subsequent analysis processes or applications.
초미세여과법(ultrafiltration)은 초원심분리법과 함께 사용되어, 엑소좀, 세포외소포체 또는 세포 유래 베시클의 순도를 높일 수 있지만, 엑소좀, 세포외소포체 또는 세포 유래 베시클이 필터에 달라붙어 분리 후의 수율이 낮다는 문제점이 있다.Ultrafiltration can be used in conjunction with ultracentrifugation to increase the purity of exosomes, extracellular vesicles, or cell-derived vesicles, but the exosomes, extracellular vesicles or cell-derived vesicles adhere to the filter and separate There is a problem in that the subsequent yield is low.
또한, 면역친화성 분리법은 항체를 엑소좀, 세포외소포체 또는 세포 유래 베시클에 붙여서 분리하는 방법으로 특이도가 높은 장점이 있지만, 항체를 만드는 과정과 분리 후 항체를 제거하는 과정이 필요하고 비용이 많이 드는 단점이 있으며 스케일-업(scale-up)에 부적합한 방식이다. In addition, the immunoaffinity separation method has the advantage of high specificity by attaching the antibody to exosomes, extracellular vesicles, or cell-derived vesicles. This has the disadvantage of being expensive, and it is an unsuitable method for scale-up.
한편, 최근에는 엑소좀을 분리하는 방법으로 침전법(exosome precipitation), 총 엑소좀 추출 키트(total exosome isolation kit), 또는 폴리머 기반 침전법(polymer based precipitation) 등의 다양한 엑소좀 분리 키트가 상업적으로 판매되고 있으나, 사용이 간편한 대신 시약값이 고가여서 실험실 수준에서 엑소좀 또는 세포외소포체를 분리하기 위해 사용될 수는 있어도 대량으로 엑소좀 또는 세포외소포체를 분리 및 정제하기에는 적합하지 않은 문제가 있다.On the other hand, recently, as a method of isolating exosomes, various exosome separation kits such as exosome precipitation, total exosome isolation kit, or polymer based precipitation are commercially available. Although it is sold, it is easy to use, but the reagent is expensive, so it can be used to isolate exosomes or extracellular vesicles at the laboratory level, but there is a problem that is not suitable for isolating and purifying exosomes or extracellular vesicles in large quantities.
무엇보다도 엑소좀, 세포외소포체 또는 세포 유래 베시클의 분리, 정제과정에서 문제가 되는 것은 수율이 낮거나, 시간이 많이 소모되고 번거로우며, 비용이 많이 소모된다는 것이다. 또한, 순도를 높이기 위해 개발된 기존의 분리 방법은 스케일-업(scale-up)을 어렵게 하고 GMP(Good Manufacturing Practice)에 부적합한 문제가 있다. Above all, a problem in the separation and purification process of exosomes, extracellular vesicles, or cell-derived vesicles is that the yield is low, time-consuming, cumbersome, and costly. In addition, the conventional separation method developed to increase the purity is difficult to scale-up (scale-up), there is a problem that is unsuitable for GMP (Good Manufacturing Practice).
따라서, 본 발명이 속한 기술분야에서는 엑소좀 또는 세포외소포체, 특히 세포 유래 베시클을 효율적으로 분리 및 정제할 수 있는 기술에 대한 요구가 꾸준히 계속되고 있다.Therefore, in the technical field to which the present invention belongs, there is a steady demand for a technology capable of efficiently separating and purifying exosomes or extracellular vesicles, particularly cell-derived vesicles.
본 발명은 세포 유래 베시클이 포함된 시료로부터 세포 유래 베시클을 분리, 정제하는 방법에 대하여 연구하던 중, 세포 유래 베시클을 포함하는 시료를 특정 조건의 십자류여과(tangential flow filtration, TFF)방식으로 분리, 정제하면 세포 유래 베시클이 고수율, 고순도로 분리, 정제되는 것을 확인하였는바, 본 발명을 완성하였다.In the present invention, while research on a method for separating and purifying cell-derived vesicles from a sample containing cell-derived vesicles, a sample containing cell-derived vesicles was subjected to tangential flow filtration (TFF) under specific conditions. After separation and purification in this way, it was confirmed that the cell-derived vesicles were separated and purified in high yield and high purity, thereby completing the present invention.
따라서, 본 발명의 목적은 세포 유래 베시클이 포함된 시료로부터 세포 유래 베시클을 효율적으로 분리, 정제하는 방법을 제공하는 것이다.Accordingly, an object of the present invention is to provide a method for efficiently separating and purifying cell-derived vesicles from a sample containing cell-derived vesicles.
상기 목적을 달성하기 위하여, 본 발명은 a) 세포 유래 베시클을 포함하는 시료를 60 내지 120 ml/분의 유량(flow rate) 및 0.1 내지 0.3 bar의 막간차압(transmembrane pressure)을 유지하며, 분자량 차단(molecular weight cutoff, MWCO)이 500 내지 1,000kDa인 십자류여과(tangential flow filtration, TFF) 필터를 사용하여, 십자류여과를 실시하는 단계; 및 b) 공극의 크기가 0.2 내지 0.5μm인 필터를 이용하여 여과하는 단계를 포함하고, 상기 a) 단계의 십자류여과는 1) 상기 시료의 초기 부피의 20 내지 40 %가 되도록 한외여과를 통해 농축하는 단계; 2) 농축이 끝난 부피 기준 4 내지 8배의 부피를 갖는 완충용액을 이용하여 탈염과 버퍼교환을 수행하는 단계; 및 3) 상기 시료의 초기 부피의 3 내지 7 %가 되도록 한외여과를 통해 재농축하는 단계를 포함하는 것인, 세포 유래 베시클의 분리 및 정제 방법을 제공한다.In order to achieve the above object, the present invention maintains a) a sample containing a cell-derived vesicle at a flow rate of 60 to 120 ml/min and a transmembrane pressure of 0.1 to 0.3 bar, molecular weight Using a tangential flow filtration (TFF) filter having a molecular weight cutoff (MWCO) of 500 to 1,000 kDa, performing cross flow filtration; and b) filtering using a filter having a pore size of 0.2 to 0.5 μm, wherein the cross-flow filtration in step a) is 1) through ultrafiltration to 20 to 40% of the initial volume of the sample. concentrating; 2) performing desalting and buffer exchange using a buffer solution having a volume of 4 to 8 times the volume of the concentrated volume; And 3) it provides a method for separating and purifying cell-derived vesicles, comprising the step of re-concentrating through ultrafiltration to 3 to 7% of the initial volume of the sample.
또한, 본 발명은 상기 방법에 의해 분리 및 정제된 세포 유래 베시클을 제공한다.In addition, the present invention provides a cell-derived vesicle isolated and purified by the above method.
본 발명에 따른 세포 유래 베시클 분리 및 정제방법을 이용하면, 고수율, 고순도로 세포 유래 베시클을 분리 및 정제할 수 있다. Using the cell-derived vesicle separation and purification method according to the present invention, cell-derived vesicles can be isolated and purified with high yield and high purity.
도 1은 중간엽 줄기세포 유래 베시클의 분리 및 정제 과정을 도식화하여 나타낸 도이다.1 is a diagram schematically showing the separation and purification process of mesenchymal stem cell-derived vesicles.
도 2는 십자류여과시의 막간차압을 0.1, 0.2, 0.5 bar로 달리하고, 각각의 정제 단계별 정제 전 세포 유래 베시클(Crude CDV) 대비 입자수, 단백질, DNA의 비율을 비교하여 나타낸 도이다.2 is a diagram showing the ratio of the number of particles, protein, and DNA compared to the cell-derived vesicle (Crude CDV) before purification at each purification step by varying the transmembrane pressure during cross flow filtration to 0.1, 0.2, and 0.5 bar. .
도 3은 십자류여과시의 유량을 60, 80, 100, 120 mL/분으로 달리하고, 각각의 정제 단계별 정제 전 세포 유래 베시클(Crude CDV) 대비 입자수, 단백질, DNA의 비율을 비교하여 나타낸 도이다.3 is a flow rate of 60, 80, 100, and 120 mL/min during cross-flow filtration, and the ratio of the number of particles, protein, and DNA compared to the cell-derived vesicle (Crude CDV) before purification for each purification step is compared. is the diagram shown.
도 4는 십자류여과시 TFF 필터의 MWCO를 750kDa와 100kDa으로 달리하고, 순도, 각각의 정제 단계별 정제 전 세포 유래 베시클(Crude CDV) 대비 입자수, 단백질, DNA의 비율을 비교하여 나타낸 도이다.4 is a diagram showing the ratio of the number of particles, protein, and DNA compared to the cell-derived vesicle (Crude CDV) prior to purification by varying the MWCO of the TFF filter to 750 kDa and 100 kDa during cross flow filtration, and for each purification step. .
도 5는 한외여과(Ultrafiltration)과정의 희석배수를 14배, 20배, 22배, 23배로 달리하고, 수율(particle/cell) 및 순도(particle/ug)를 비교한 도이다.5 is a diagram comparing the yield (particle/cell) and purity (particle/ug) by varying the dilution factor of the ultrafiltration process by 14 times, 20 times, 22 times, and 23 times.
도 6은 정용여과(diafiltration)과정에서 사용하는 완충용액의 부피를 1 내지 6배로 달리하고, 각각 정제 단계별 정제 전 세포 유래 베시클(Crude CDV) 대비 입자수, 단백질, DNA의 비율을 비교한 도이다.6 is a diagram comparing the ratio of the number of particles, protein, and DNA to cell-derived vesicles (Crude CDV) prior to purification in each purification step by varying the volume of the buffer solution used in the diafiltration process by 1 to 6 times; am.
도 7은 여과필터 공극의 크기를 0.45um와 0.22um로 달리하고, 각각의 여과 전후의 세포 유래 베시클 입자수, 단백질 수, 크기, PDI(polydispersity index, 다분산지수)를 비교한 도이다.7 is a diagram comparing the number of cell-derived vesicle particles, the number of proteins, the size, and the polydispersity index (PDI) before and after each filtration by varying the size of the pores of the filtration filter to 0.45um and 0.22um.
도 8은 실시예 2의 방법으로 중간엽 줄기세포 유래 베시클을 분리 및 정제한 경우, 수율을 나타낸 도이다.8 is a diagram showing the yield when the mesenchymal stem cell-derived vesicles are isolated and purified by the method of Example 2.
도 9는 실시예 2의 방법으로 중간엽 줄기세포 유래 베시클을 분리 및 정제한 경우, 순도를 나타낸 도이다.9 is a diagram showing the purity when the mesenchymal stem cell-derived vesicles are isolated and purified by the method of Example 2.
도 10은 다양한 정제 방법 gUC, UC 와 본 발명의 정제방법(TFF) 에 따른 세포 유래 베시클의 수득률 및 순도를 비교한 결과를 나타낸 도이다. 10 is a diagram showing the results of comparing the yield and purity of cell-derived vesicles according to various purification methods gUC and UC and the purification method (TFF) of the present invention.
본 발명은 a) 세포 유래 베시클을 포함하는 시료를 60 내지 120 ml/분의 유량(flow rate) 및 0.1 내지 0.3 bar의 막간차압(transmembrane pressure)을 유지하며, 분자량 차단(molecular weight cutoff, MWCO)이 500 내지 1,000kDa인 십자류여과(tangential flow filtration, TFF) 필터를 사용하여, 십자류여과를 실시하는 단계; 및 b) 공극의 크기가 0.2 내지 0.5μm인 필터를 이용하여 여과하는 단계를 포함하고, 상기 a) 단계의 십자류여과는 1) 상기 시료의 초기 부피의 20 내지 40 %가 되도록 한외여과를 통해 농축하는 단계; 2) 농축이 끝난 부피 기준 4 내지 8배의 부피를 갖는 완충용액을 이용하여 탈염과 버퍼교환을 수행하는 단계; 및 3) 상기 시료의 초기 부피의 3 내지 7 %가 되도록 한외여과를 통해 재농축하는 단계를 포함하는 것인, 세포 유래 베시클의 분리 및 정제 방법을 제공한다.The present invention a) maintains a flow rate of 60 to 120 ml/min and a transmembrane pressure of 0.1 to 0.3 bar for a sample containing a cell-derived vesicle, and molecular weight cutoff (MWCO) ) using a 500 to 1,000 kDa cross-flow filtration (tangential flow filtration, TFF) filter, performing cross-flow filtration; and b) filtering using a filter having a pore size of 0.2 to 0.5 μm, wherein the cross-flow filtration in step a) is 1) through ultrafiltration to 20 to 40% of the initial volume of the sample. concentrating; 2) performing desalting and buffer exchange using a buffer solution having a volume of 4 to 8 times the volume of the concentrated volume; And 3) it provides a method for separating and purifying cell-derived vesicles, comprising the step of re-concentrating through ultrafiltration to 3 to 7% of the initial volume of the sample.
본 발명에 있어서, 세포 유래 베시클은 유핵세포에서 인위적으로 제조된 베시클을 말하며, 거의 모든 종류의 세포에서 세포막으로부터 유리되어, 세포막의 구조인 이중 인지질(phospholipid) 막 형태를 가지고 있는 것을 말한다. 본 발명의 세포 유래 베시클은 마이크로미터 크기, 예컨대 0.03~1㎛를 가질 수 있다. In the present invention, the cell-derived vesicle refers to a vesicle artificially manufactured in a nucleated cell, is separated from the cell membrane in almost all types of cells, and has a double phospholipid membrane, which is the structure of the cell membrane. The cell-derived vesicle of the present invention may have a micrometer size, for example, 0.03 to 1 μm.
본 발명의 세포 유래 베시클은 자연적으로 분비되는 베시클과 구별되며, 세포를 포함하는 시료를 미세공극에 압출하여 제조되는 것 일 수 있으며, 바람직하게는 미세공극의 크기가 큰 것으로부터 미세공극의 크기가 작은 것으로 순차적으로 압출하여 제조되는 것 일 수 있으며, 바람직하게는 미세공극의 크기가 9 내지 11μm, 2 내지 4 μm 및 0.6 내지 0.2 μm인 멤브레인 필터, 더욱 바람직한 일 구현예로는 10 μm, 3 μm 및 0.4μm 인 멤브레인 필터에 순차적으로 압출하여 제조되는 것 일 수 있다. The cell-derived vesicle of the present invention is distinguished from the naturally secreted vesicle, and may be prepared by extruding a sample containing cells into micropores, and preferably, the micropore size is larger than the micropore size. It may be manufactured by sequential extrusion with a small size, preferably a membrane filter having a micropore size of 9 to 11 μm, 2 to 4 μm, and 0.6 to 0.2 μm, more preferably 10 μm, It may be manufactured by sequentially extruding a membrane filter of 3 μm and 0.4 μm.
본 발명에 있어서, 상기 세포 유래 베시클 제조를 위한 세포는 유핵세포라면 제한없이 포함되는 것 일 수 있으나, 바람직하게는 줄기세포, 선포세포, 근상피세포, 적혈구, 단핵구, 수지상 세포, 자연살해 세포 및 혈소판으로 이루어진 군에서 선택된 어느 하나 이상일 수 있다. 상기 줄기세포는 중간엽 줄기세포, 유도만능줄기세포, 배아줄기세포 및 침샘 줄기세포로 이루어진 군에서 선택된 어느 하나 이상일 수 있고, 더욱 바람직하게는 중간엽 줄기세포, 더더욱 바람직하게 상기 중간엽 줄기세포는 지방, 골수, 제대 또는 제대혈 유래 중간엽 줄기세포일 수 있다.In the present invention, the cells for preparing the cell-derived vesicles may include without limitation as long as they are nucleated cells, but preferably stem cells, acinar cells, myoepithelial cells, red blood cells, monocytes, dendritic cells, and natural killer cells. And it may be any one or more selected from the group consisting of platelets. The stem cells may be any one or more selected from the group consisting of mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells and salivary gland stem cells, more preferably mesenchymal stem cells, even more preferably the mesenchymal stem cells It may be adipose, bone marrow, umbilical cord or umbilical cord blood-derived mesenchymal stem cells.
본 발명의 ‘베시클'은 유래한 세포의 세포막 성분으로 이루어진 지질 이중막에 의해 내부와 외부가 구분되며, 세포의 세포막 지질과 세포막 단백질, 핵산 및 세포 성분 등을 가지고 있으며, 원래 세포보다 크기가 작은 것을 의미하지만, 이에 제한되는 것은 아니다.The 'vesicle' of the present invention is separated from the inside and outside by a lipid double membrane composed of the cell membrane component of the derived cell, and has cell membrane lipids, cell membrane proteins, nucleic acids and cell components of the cell, and is larger in size than the original cell. means small, but not limited thereto.
본 발명에 있어서, 상기 탈염과 버퍼교환은 연속적 또는 비-연속적으로 수행되는 것 일 수 있으나, 바람직하게는 연속적으로 수행되는 것 일 수 있다. In the present invention, the desalting and buffer exchange may be carried out continuously or non-continuously, but preferably may be carried out continuously.
본 발명에 있어서, 상기 용어 십자류여과(tangential flow filtration, TFF)는 시료가 여과되는 방향과 시료가 공급되는 방향이 직각인 여과 방식을 의미한다. In the present invention, the term tangential flow filtration (TFF) refers to a filtration method in which the direction in which the sample is filtered and the direction in which the sample is supplied are perpendicular.
본 발명에 있어서, 상기 유량(flow rate)은 단위 시간당 통과하는 유체의 부피를 의미하며, 바람직하게 상기 유량은 60 내지 120ml/분일 수 있고, 바람직하게는 70 내지 90 ml/분일 수 있고, 더욱 바람직하게는 세포 유래 베시클의 표면 단백질이 전단 응력에 취약한 특성을 고려하여 80ml/분일 수 있다. In the present invention, the flow rate (flow rate) means the volume of fluid passing per unit time, preferably the flow rate may be 60 to 120 ml / min, preferably 70 to 90 ml / min, more preferably Preferably, it may be 80 ml/min in consideration of the property that the surface protein of the cell-derived vesicle is vulnerable to shear stress.
본 발명에 있어서, 상기 막간차압(transmembrane pressure)은 여과 필터를 경계로 한 압력차이를 의미하며, 바람직하게 상기 막간차압은 0.1 내지 0.3 bar일 수 있고, 더욱 바람직하게는 0.2 bar일 수 있다. 막간차압을 0.2 bar로 하는 경우, 시료 내 존재하는 단백질 및 DNA를 가장 효과적으로 제거할 수 있다.In the present invention, the transmembrane pressure means a pressure difference with the filtration filter as a boundary, and preferably, the transmembrane pressure may be 0.1 to 0.3 bar, more preferably 0.2 bar. When the transmembrane pressure is 0.2 bar, proteins and DNA present in the sample can be most effectively removed.
본 발명에 있어서, 상기 분자량 차단(molecular weight cutoff, MWCO)은 용질의 90%가 여과 필터에 의해 유지되는 용질의 최저 분자량을 의미하며, 바람직하게 상기 분자량 차단은 500 내지 1,000kDa 일 수 있고, 바람직하게는 700 내지 800kDa일 수 있으며, 더욱 바람직하게는 750kDa일 수 있다. 분자량 차단을 750kDa으로 하는 경우, 세포 유래 베시클을 고순도로 분리 및 정제가 가능하다.In the present invention, the molecular weight cutoff (MWCO) means the lowest molecular weight of the solute in which 90% of the solute is maintained by the filtration filter, preferably the molecular weight cutoff may be 500 to 1,000 kDa, preferably Preferably, it may be 700 to 800 kDa, and more preferably 750 kDa. When the molecular weight cutoff is 750 kDa, it is possible to separate and purify cell-derived vesicles with high purity.
본 발명에 있어서, 상기 한외여과(ultrafiltration)는 아주 작은 구멍이 있는 여과막을 통해 여과하여 극히 미세한 입자를 분리하는 여과방식을 의미하며, 여과막을 통과하지 않은 용액은 농축된다. In the present invention, the ultrafiltration refers to a filtration method in which extremely fine particles are separated by filtration through a filtration membrane having very small pores, and the solution that does not pass through the filtration membrane is concentrated.
본 발명에 있어서, 상기 한외여과는 초기 부피의 20 내지 40 %가 되도록 1차 농축하고, 탈염과 버퍼교환 이 후에 초기 부피의 3 내지 7%가 되도록 2차 농축하는 것일 수 있으며, 가장 바람직하게는 초기 부피의 25 %가 되도록 1차 농축하고, 탈염과 버퍼교환 이 후에 초기 부피의 바람직하게는 4 내지 6%, 가장 바람직하게는 5 %가 되도록 2차 재농축하는 것일 수 있다. In the present invention, the ultrafiltration may be a primary concentration of 20 to 40% of the initial volume, and a secondary concentration of 3 to 7% of the initial volume after desalting and buffer exchange, most preferably The primary concentration may be 25% of the initial volume, and the secondary re-concentration may be preferably 4 to 6%, most preferably 5% of the initial volume after desalting and buffer exchange.
본 발명에 있어서, 상기 탈염과 버퍼교환은 완충용액을 이용하여 정용여과(diafiltration)하여 수행되는 것 일 수 있으며, 본 발명에 있어서, 상기 탈염과 버퍼교환은 농축이 끝난 시료의 부피를 기준으로 5 내지 7 배의 부피를 갖는 완충용액을 이용하여 정용여과하는 것 일 수 있고, 가장 바람직하게는 농축이 끝난 시료의 부피를 기준으로 6배의 부피를 갖는 완충용액을 이용하여 정용여과하는 것 일 수 있다. In the present invention, the desalting and buffer exchange may be performed by diafiltration using a buffer solution, and in the present invention, the desalting and buffer exchange are 5 based on the volume of the concentrated sample. It may be diafiltration using a buffer solution having a volume of to 7 times, and most preferably, diafiltration using a buffer solution having a volume of 6 times the volume of the concentrated sample. have.
따라서 본 발명의 2) 단계는 농축이 끝난 부피 기준 5 내지 7 배의 부피를 갖는 완충용액을 이용하여 탈염과 버퍼교환을 수행하는 단계이고, 상기 3) 단계는 상기 시료의 초기 부피의 3 내지 7%이 되도록 한외여과를 통해 재농축하는 단계일 수 있다. Therefore, step 2) of the present invention is a step of performing desalting and buffer exchange using a buffer solution having a volume 5 to 7 times the volume based on the finished volume, and step 3) is 3 to 7 of the initial volume of the sample. % may be a step of re-concentration through ultrafiltration.
본 발명에 있어서, 초기 부피의 25 %가 되도록 1차 한외여과를 수행하여 농축하고, 농축이 끝난 시료 부피를 기준으로 6배의 부피를 갖는 완충용액을 이용한 정용여과를 통해 탈염과 버퍼교환을 수행하며, 시료 초기 부피의 3 내지 7%가 되도록 2차 한외여과를 수행하여 재농축하는 경우, 80 % 이상의 단백질이 제거되며, 후술하는 바와 같이 핵산분해효소 처리시 95 %의 DNA가 제거되는 효과가 달성되는 것 일 수 있다.In the present invention, primary ultrafiltration is performed to concentrate to 25% of the initial volume, and desalting and buffer exchange are performed through diafiltration using a buffer having a volume of 6 times the volume of the sample after concentration is completed. In the case of re-concentration by performing secondary ultrafiltration to 3 to 7% of the initial volume of the sample, more than 80% of protein is removed, and 95% of DNA is removed during nuclease treatment as described below. can be achieved.
본 발명에 있어서, 상기 세포 유래 베시클의 분리 및 정제 방법은 상기 a) 단계의 십자류 여과를 실시하는 단계 이전에 핵산분해효소, Tris-HCl 및 염화마그네슘을 처리하는 단계를 더 포함하는 것 일 수 있으며, 바람직하게는 세포 유래 베시클을 포함하는 시료에 핵산분해효소, 40 내지 60 mM Tris-HCl 및 1 내지 3 mM 염화마그네슘을 시료 내 DNA량에 비례하여 처리하는 단계를 더 포함하는 것일 수 있다.In the present invention, the method for separating and purifying the cell-derived vesicle further comprises treating a nuclease, Tris-HCl and magnesium chloride before performing the cross flow filtration of step a). Preferably, the method may further comprise treating a sample containing a cell-derived vesicle with a nuclease, 40 to 60 mM Tris-HCl, and 1 to 3 mM magnesium chloride in proportion to the amount of DNA in the sample. have.
본 발명에 있어서, 상기 b) 단계의 필터는 공극의 크기가 0.3 내지 0.5μm인 것일 수 있으며, 바람직하게는 0.45μm인 것일 수 있다. 공극의 크기가 0.3 내지 0.5μm, 특히 0.45μm인 경우, 세포 유래 베시클의 입자 손실률이 현저히 적고, 세포 유래 베시클의 크기 및 균일성이 유지되는 것일 수 있다.In the present invention, the filter of step b) may have a pore size of 0.3 to 0.5 μm, preferably 0.45 μm. When the size of the pores is 0.3 to 0.5 μm, particularly 0.45 μm, the particle loss rate of the cell-derived vesicle is remarkably low, and the size and uniformity of the cell-derived vesicle may be maintained.
본 발명에 있어서, 본 발명의 세포 유래 베시클의 분리 및 정제 방법에 의해 분리 및 정제된 세포 유래 베시클은 크기가 100 내지 200nm이며, 다분산 지수(polydispersity index, PDI)가 0.2 내지 0.5, 바람직하게는 0.2 내지 0.3인 것일 수 있다.In the present invention, the cell-derived vesicles separated and purified by the cell-derived vesicle separation and purification method of the present invention have a size of 100 to 200 nm, and a polydispersity index (PDI) of 0.2 to 0.5, preferably For example, it may be 0.2 to 0.3.
본 발명의 세포 유래 베시클의 분리 및 정제 방법은 가장 바람직하게는, a) 세포 유래 베시클을 포함하는 시료에 핵산분해효소, 50 mM Tris-HCl 및 2 mM 염화마그네슘을 시료 내 DNA량에 비례하여 처리하는 단계; b) 20 내지 25℃인 실온에서 10분간 3000 x G 조건으로 원심분리하는 단계; c) 세포 유래 베시클을 포함하는 시료를 80 ml/분의 유량(flow rate) 및 0.2 bar의 막간차압(transmembrane pressure)을 유지하며, 분자량 차단(molecular weight cutoff, MWCO)이 750kDa인 십자류여과(tangential flow filtration, TFF) 필터를 사용하여, 십자류여과를 실시하는 단계; d) 20 내지 25℃인 실온에서 10분간 3000 x G 조건으로 원심분리하는 단계; 및 e) 공극의 크기가 0.45μm인 필터를 이용하여 여과하는 단계를 포함하고, 상기 c) 단계의 십자류여과는 1) 상기 시료의 초기 부피의 25 %가 되도록 한외여과를 통해 농축하는 단계; 2) 농축이 끝난 부피 기준 6 배의 부피를 갖는 완충용액을 이용하여 탈염과 버퍼교환을 수행하는 단계; 및 3) 상기 시료의 초기 부피의 5 %가 되도록 한외여과를 통해 재농축하는 단계를 포함하여 수행하는 것 일 수 있으며, 이러한 단계로 세포 유래 베시클을 분리 및 정제하는 경우, 세포 유래 베시클을 고수율, 고순도로 분리, 정제할 수 있는 것 일 수 있다.The method for isolating and purifying cell-derived vesicles of the present invention most preferably comprises: a) adding nuclease, 50 mM Tris-HCl and 2 mM magnesium chloride to a sample containing cell-derived vesicles in proportion to the amount of DNA in the sample to process; b) centrifugation at 3000 x G conditions for 10 minutes at a room temperature of 20 to 25 °C; c) cross flow filtration of a sample containing cell-derived vesicles with a flow rate of 80 ml/min and a transmembrane pressure of 0.2 bar, and a molecular weight cutoff (MWCO) of 750 kDa (tangential flow filtration, TFF) using a filter, performing cross-flow filtration; d) centrifugation at 3000 x G conditions for 10 minutes at room temperature of 20 to 25 °C; and e) filtering using a filter having a pore size of 0.45 μm, wherein the cross-flow filtration in step c) includes 1) concentrating through ultrafiltration to 25% of the initial volume of the sample; 2) performing desalting and buffer exchange using a buffer solution having a volume of 6 times the volume based on the concentrated volume; and 3) re-concentration through ultrafiltration to 5% of the initial volume of the sample. It may be one that can be separated and purified with high yield and high purity.
또한, 본 발명은 상기 방법에 의해 분리 및 정제된 세포 유래 베시클을 제공한다.In addition, the present invention provides a cell-derived vesicle isolated and purified by the above method.
본 발명에 있어서, 상기 세포 유래 베시클은 크기가 100 내지 200nm이며, 다분산 지수(polydispersity index, PDI)가 0.2 내지 0.5, 바람직하게는 0.2 내지 0.3인 것일 수 있다.In the present invention, the cell-derived vesicle may have a size of 100 to 200 nm, and a polydispersity index (PDI) of 0.2 to 0.5, preferably 0.2 to 0.3.
중복되는 내용은 본 명세서의 복잡성을 고려하여 생략하며, 본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Duplicate content is omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification have the meanings commonly used in the art to which the present invention pertains.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited by the following examples.
실시예 1. 정제 전 중간엽 줄기세포 유래 베시클 현탁액의 제조Example 1. Preparation of mesenchymal stem cell-derived vesicle suspension before purification
제대혈 중간엽 줄기세포(UC MSC)로부터 압출법을 통하여 베시클 현탁액을 제조하였다. 줄기세포 complete growth medium에서 배양된 중간엽 줄기세포를 인산완충식염수(phosphate buffered saline; PBS)로 세척하고, 세척한 줄기세포를 0.25 내지 1 x 10 6 cells/ml의 농도로 하여 PBS에 재부유(resuspension)시켰다. 세포 압출기(extruder)로 상기 현탁 용액을 미세공극의 크기가 10μm인 멤브레인 필터(membrane filter)에 통과시킨 후, 미세공극의 크기가 3μm인 멤브레인 필터에 통과시키고, 이어서 미세공극의 크기가 0.4μm인 멤브레인 필터에 통과시켜, 중간엽 줄기세포 유래 베시클(Crude UCMSC-CDV) 현탁액을 제조하였다.A vesicle suspension was prepared from umbilical cord blood mesenchymal stem cells (UC MSC) by extrusion. Mesenchymal stem cells cultured in stem cell complete growth medium are washed with phosphate buffered saline (PBS), and the washed stem cells are resuspended in PBS at a concentration of 0.25 to 1 x 10 6 cells/ml ( resuspension). The suspension solution was passed through a membrane filter having a micropore size of 10 μm using a cell extruder, and then passed through a membrane filter having a micropore size of 3 μm, followed by a micropore size of 0.4 μm. By passing through a membrane filter, a suspension of mesenchymal stem cell-derived vesicles (Crude UCMSC-CDV) was prepared.
실시예 2. 중간엽 줄기세포 유래 베시클의 분리 및 정제 방법Example 2. Isolation and purification method of mesenchymal stem cell-derived vesicles
실시예 1과 같은 방법으로, 세포 압출기를 통해 생산한 중간엽 줄기세포 유래 베지클(Crude UCMSC-CDV)의 분리 및 정제를 위해, 십자류여과(Tangential Flow Filtration, TFF)를 이용하여 100~200nm의 크기를 가지며 PDI가 0.2 내지 0.3의 균일도를 갖도록 중간엽 줄기세포 유래 베지클을 정제하였다. 구체적으로, Spectrum® KR2i TFF Systems (Repligen), hollow fiber 필터 (Repligen, 750kDa, 0.01m 2)를 이용하여 중간엽 줄기세포 유래 베시클을 분리 및 정제하였다. 실시예 1과 같은 방법으로 세포 압출기에서 추출한 베시클 현탁액의 DNA량에 비례하여 벤조네이즈 핵산분해효소 (benzonase nuclease, Merck, 71206-3), 50mM Tris-HCl (Teknova, 50-843-335), 2mM 염화마그네슘 (Magnesium chloride, SIGMA-ALDRICH, M1028-100mL)을 90분, 37℃ 조건에서 처리하였다. TFF 방법을 이용하여 베지클을 분리/정제하기 전에 실온에서 10분간 3000 x G 조건으로 원심분리하여 베지클 현탁액 내 응집체를 제거하였다. 0.2 bar의 막간차압(transmembrane pressure)을 유지하며 80 mL/분의 유량(flow rate)으로 현탁액을 농축하고 불순물을 걸러내었다. 한외여과(Ultrafiltration)과정에서 현탁액의 부피가 1/4 정도의 부피가 될 때까지 농축하고, 이어서 정용여과(diafiltration)과정에서 탈염과 버퍼교환을 통해 불순물을 제거하였다. 탈염과 버퍼교환은 연속적으로 수행하였으며, 농축이 끝난 부피에 대하여 적어도 6배의 부피를 갖는 완충용액(PBS)을 이용하였다. 마지막으로 한외여과(Ultrafiltration)과정을 다시 실시하여 현탁액의 부피가 시작 부피(정제 전의 베지클 현탁액 부피)의 1/20가 될 때까지 농축하였다. 분리/정제된 중간엽 줄기세포 유래 베지클을 실온에서 10분간 3000 x G 조건으로 원심분리하여 응집체를 제거하고 0.45um 시린지 필터(pall, 4614)에 걸러, 최종적으로 중간엽 줄기세포 유래 베지클을 획득하였다. 이후, DLS, NTA, 측정을 통해 정제된 중간엽 줄기세포 유래 베지클의 물성을 분석하고 단백질, DNA, 벤조네이즈의 정량을 통해 정제 전 대비 불순물 제거율을 확인하였다. 상기와 같은 중간엽 줄기세포 유래 베시클의 분리 및 정제 과정을 도식화하여 도 1에 나타내었다. In the same manner as in Example 1, for separation and purification of mesenchymal stem cell-derived vesicles (Crude UCMSC-CDV) produced through a cell extruder, 100-200 nm using Tangential Flow Filtration (TFF) Mesenchymal stem cell-derived vesicles were purified to have a size of and PDI to have a uniformity of 0.2 to 0.3. Specifically, mesenchymal stem cell-derived vesicles were isolated and purified using Spectrum® KR2i TFF Systems (Repligen), a hollow fiber filter (Repligen, 750 kDa, 0.01 m 2 ). In proportion to the amount of DNA in the vesicle suspension extracted from the cell extruder in the same manner as in Example 1, benzonase nuclease (Merck, 71206-3), 50 mM Tris-HCl (Teknova, 50-843-335), 2mM magnesium chloride (Magnesium chloride, SIGMA-ALDRICH, M1028-100mL) was treated for 90 minutes, 37 ℃ condition. Before separating/purifying the vesicles using the TFF method, the aggregates in the vesicle suspension were removed by centrifugation at 3000 x G conditions for 10 minutes at room temperature. The suspension was concentrated at a flow rate of 80 mL/min while maintaining a transmembrane pressure of 0.2 bar, and impurities were filtered off. In the process of ultrafiltration, the suspension was concentrated until the volume was about 1/4 of the volume, and then impurities were removed through desalting and buffer exchange in the diafiltration process. Desalting and buffer exchange were performed continuously, and a buffer solution (PBS) having at least 6 times the volume of the concentrated volume was used. Finally, the ultrafiltration process was performed again and the suspension was concentrated until the volume of the suspension became 1/20 of the starting volume (the volume of the vesicle suspension before purification). The separated/purified mesenchymal stem cell-derived vesicles were centrifuged at room temperature for 10 minutes at 3000 x G to remove aggregates, filtered through a 0.45um syringe filter (pall, 4614), and finally mesenchymal stem cell-derived vesicles were removed. obtained. Thereafter, the physical properties of the mesenchymal stem cell-derived vesicles purified through DLS, NTA, and measurement were analyzed, and the removal rate of impurities compared to before purification was confirmed through quantification of protein, DNA, and benzonase. The process of separation and purification of the mesenchymal stem cell-derived vesicles as described above is schematically shown in FIG. 1 .
실시예 3.Example 3. 중간엽 줄기세포 유래 베시클의 분리 및 정제 조건 선정Selection of conditions for isolation and purification of mesenchymal stem cell-derived vesicles
3.1 막간차압의 선정3.1 Selection of transmembrane pressure
정제 후 입자 회수율을 기반으로 DNA 및 비특이 단백질의 제거 효율이 높은 유량 및 막간차압의 조건을 선정하였다. 막간차압을 선정하기 위해, 실시예 2의 방법에서 막간차압을 0.1, 0.2, 0.5 bar로 달리하고, 각각의 정제 단계별 정제 전 세포 유래 베시클(Crude CDV) 대비 입자수, 단백질, DNA의 비율을 하기와 같이 측정하였다.Based on the particle recovery rate after purification, the conditions of high flow rate and transmembrane pressure with high DNA and non-specific protein removal efficiency were selected. In order to select the transmembrane pressure, in the method of Example 2, the transmembrane pressure was changed to 0.1, 0.2, and 0.5 bar, and the ratio of the number of particles, protein, and DNA to cell-derived vesicle (Crude CDV) before purification for each purification step was calculated. Measurements were made as follows.
세포 유래 베시클(Crude CDV) 대비 입자수는 NTA (Nanoparticle Tracking Analysis; Nanosight NS300, Malvern Panalytical, Westborough, MA, USA)로 측정하였다. The number of particles compared to cell-derived vesicles (Crude CDV) was measured by NTA (Nanoparticle Tracking Analysis; Nanosight NS300, Malvern Panalytical, Westborough, MA, USA).
단백질 농도는 Qubit ™ 4 플루오로미터 (Thermo Scientific, Waltham, MA, USA)와 Qubit™ 단백질 어세이 키트(Protein Assay Kit, Thermo Fisher scientific)를 이용하여 측정하였다. 190uL의 워킹 솔루션을 각각 10uL의 샘플 및 표준용액과 혼합하여 샘플 및 표준용액을 준비하였다. 각 혼합물을 2-3 초 동안 볼텍싱하고, 실온에서 15 분 동안 인큐베이션하였다. 이 후 표준용액 및 샘플을 순차적으로 장비에 넣고 샘플 내 단백질 농도를 측정하였다.Protein concentration was measured using a Qubit™ 4 fluorometer (Thermo Scientific, Waltham, MA, USA) and a Qubit™ Protein Assay Kit (Thermo Fisher scientific). Samples and standard solutions were prepared by mixing 190 uL of working solution with 10 uL of sample and standard solution, respectively. Each mixture was vortexed for 2-3 seconds and incubated for 15 minutes at room temperature. After that, the standard solution and the sample were sequentially put into the equipment, and the protein concentration in the sample was measured.
DNA의 비율은 Qubit ™ 4 플루오로미터 (Thermo Scientific, Waltham, MA, USA)와 Qubit™ HS dsDNA 어세이 키트 (Thermo Fisher scientific)를 사용하여 측정하였다. 190uL의 워킹 솔루션을 각각 10uL의 샘플 및 표준용액과 혼합하여 샘플 및 표준용액을 준비하였다. 각 혼합물을 2-3 초 동안 볼텍싱한 다음 실온에서 2 분 동안 인큐베이션하였다. 이후 표준용액 및 샘플을 순차적으로 장비에 넣고 샘플의 DNA 농도를 측정하였다.The proportion of DNA was measured using a Qubit™ 4 fluorometer (Thermo Scientific, Waltham, MA, USA) and a Qubit™ HS dsDNA assay kit (Thermo Fisher scientific). Samples and standard solutions were prepared by mixing 190 uL of working solution with 10 uL of sample and standard solution, respectively. Each mixture was vortexed for 2-3 seconds and then incubated for 2 minutes at room temperature. Then, the standard solution and the sample were sequentially put into the equipment and the DNA concentration of the sample was measured.
상기와 같은 방법으로 각각의 정제 단계별 정제 전 세포 유래 베시클(Crude CDV) 대비 입자수, 단백질, DNA의 비율을 비교한 결과를 도 2에 나타내었다.The results of comparing the ratios of the number of particles, protein, and DNA to cell-derived vesicles (Crude CDV) before purification for each purification step in the same manner as above are shown in FIG. 2 .
도 2에 나타낸 바와 같이, 막에 가해지는 압력인 막간차압이 증가할수록 베시클 입자수, 단백질, DNA의 회수율이 감소하는 경향을 확인하였다. 또한, 막간차압이 0.1 및 0.2 bar인 경우 유사한 입자회수율을 보이지만, 단백질 및 DNA의 제거율은 0.2 bar에서 더 높은 것을 확인하였다. 이를 통해 0.2 bar가 가장 우수한 막간차압에 해당하는 것을 확인하였다. As shown in FIG. 2 , as the transmembrane pressure, which is the pressure applied to the membrane, increased, it was confirmed that the number of vesicle particles, the recovery rate of protein, and DNA decreased. In addition, when the transmembrane pressure was 0.1 and 0.2 bar, it was confirmed that the particle recovery rate was similar, but the protein and DNA removal rate was higher at 0.2 bar. Through this, it was confirmed that 0.2 bar corresponds to the best transmembrane pressure.
3.2 유량(Flow rate)의 선정3.2 Flow rate selection
실시예 3.1과 같이, 유량을 60, 80, 100, 120 mL/분으로 달리하고, 각각의 정제 단계별 정제 전 세포 유래 베시클(Crude CDV) 대비 입자수, 단백질, DNA의 비율을 비교하였으며, 그 결과를 도 3에 나타내었다.As in Example 3.1, the flow rate was changed to 60, 80, 100, and 120 mL/min, and the ratio of the number of particles, protein, and DNA to the cell-derived vesicle (Crude CDV) before purification for each purification step was compared, and the The results are shown in FIG. 3 .
도 3에 나타낸 바와 같이, 유량이 60mL/분인 경우, 베시클의 회수율이 가장 낮은 점을 제외하고 flow rate에 따른 유의미한 차이는 확인되지 않았다. 따라서 세포 유래 베시클의 표면 단백질이 전단 응력(shear stress)에 취약한 특성을 고려하여 유량을 80mL/분으로 선정하였다.As shown in FIG. 3 , when the flow rate was 60 mL/min, no significant difference was observed depending on the flow rate, except that the recovery rate of the vesicle was the lowest. Therefore, a flow rate of 80 mL/min was selected in consideration of the vulnerability of the surface protein of the cell-derived vesicle to shear stress.
3.3 MWCO(molecular weight cut off)의 선정3.3 Selection of molecular weight cut off (MWCO)
TFF 필터의 가장 우수한 MWCO 값을 선정하기 위한 실험을 실시하였다. 실시예 2의 방법에서 TFF 필터의 MWCO를 750kDa와 100kDa으로 달리하고, "최종 분획물에 포함되어 있는 단백질 단위 μg 당 베지클의 입자수"로 계산한 순도(purity)와 각각의 정제 단계별 정제 전 세포 유래 베시클(Crude CDV) 대비 입자수, 단백질, DNA의 비율을 비교하였으며, 그 결과를 도 4에 나타내었다.An experiment was conducted to select the best MWCO value of the TFF filter. In the method of Example 2, the MWCO of the TFF filter was changed to 750 kDa and 100 kDa, and the purity calculated as “the number of vesicle particles per μg of protein unit contained in the final fraction” and cells before purification for each purification step The ratio of the number of particles, protein, and DNA to the derived vesicle (Crude CDV) was compared, and the results are shown in FIG. 4 .
도 4에 나타낸 바와 같이, TFF 필터의 MWCO를 750kDa와 100kDa으로 각각 비교한 결과, MWCO를 750kDa로 분리 정제한 경우, 100kDa으로 분리 정제한 경우보다 세포 유래 베시클의 순도(purity)가 2 내지 3배 증가하는 것을 확인하여 작은 단백질을 제거하여 세포 유래 베시클의 순도를 향상시키는데 750kDa이 효과적인 것을 확인하였다.As shown in FIG. 4 , as a result of comparing the MWCO of the TFF filter at 750 kDa and 100 kDa, respectively, when the MWCO was separated and purified at 750 kDa, the purity of the cell-derived vesicle was 2 to 3 compared to the case where the MWCO was separated and purified at 100 kDa. It was confirmed that 750 kDa is effective in improving the purity of cell-derived vesicles by removing small proteins by confirming the fold increase.
3.4 한외여과(ultrafiltration) 농축배수 및 정용여과(diafiltration) 과정에서 사용하는 완충용액의 부피 선정3.4 Selection of the volume of buffer solution used in ultrafiltration concentrated drainage and diafiltration process
세포 유래 베시클 정제 및 분리에서 우수한 정제효과를 나타내는 한외여과 농축배수를 선정하였다. 실시예 2의 방법에서 한외여과(Ultrafiltration)과정의 희석배수를 14배, 20배, 22배, 23배로 달리하고, 수율(particle/cell) 및 순도(particle/ug)를 비교하였으며, 그 결과를 도 5에 나타내었다.An ultrafiltration concentrated drainage that exhibits an excellent purification effect in cell-derived vesicle purification and separation was selected. In the method of Example 2, the dilution factor of the ultrafiltration process was changed to 14 times, 20 times, 22 times, and 23 times, and the yield (particle/cell) and purity (particle/ug) were compared, and the results were compared 5 is shown.
도 5에 나타낸 바와 같이, 한외여과의 농축 배수에 따른 유의미한 차이는 확인되지 않았다.As shown in FIG. 5 , a significant difference according to the concentration drainage of ultrafiltration was not confirmed.
세포 유래 베시클 정제 및 분리방법에서 우수한 탈염 및 버퍼교환 효과를 나타내는 정용여과 과정에서 사용하는 완충용액의 부피를 선정하였다. 농축이 끝난 시료 부피 기준 1 내지 6배의 부피로 완충용액의 부피를 달리하여 탈염 및 버퍼교환을 실시하고, 각각 정제 단계별 정제 전 세포 유래 베시클(Crude CDV) 대비 입자수, 단백질, DNA의 비율을 비교하였으며, 그 결과를 도 6에 나타내었다.The volume of the buffer solution used in the diafiltration process showing excellent desalting and buffer exchange effects in the cell-derived vesicle purification and separation method was selected. Desalting and buffer exchange are performed by varying the volume of the buffer solution by 1 to 6 times the volume of the concentrated sample, and the ratio of the number of particles, protein, and DNA to cell-derived vesicles (Crude CDV) before purification for each purification step. were compared, and the results are shown in FIG. 6 .
도 6에 나타낸 바와 같이, 정용여과 과정에서 사용되는 완충용액의 부피가 증가함에 따라, 단백질 및 DNA의 제거율이 증가하는 것을 확인하였다.As shown in FIG. 6 , as the volume of the buffer solution used in the diafiltration process increased, it was confirmed that the removal rate of proteins and DNA increased.
본 실시예의 결과를 토대로, 단백질이 80% 이상 제거되고, 벤조네이즈 핵산분해효소의 사용으로 DNA의 95%이상이 제거되는 조건인 한외여과 농축 배수 20배와 정용여과 완충용액의 부피 6배를 선정하였다.Based on the results of this example, 20 times the concentration of ultrafiltration and 6 times the volume of the diafiltration buffer, which are conditions in which 80% or more of protein is removed and 95% or more of DNA is removed by the use of benzonase nuclease, were selected. did.
3.5 여과필터의 공극 크기(pore size) 선정3.5 Selection of the pore size of the filtration filter
세포 유래 베시클 정제 및 분리에서 우수한 정제효과를 나타내는 여과필터 공극의 크기를 선정하였다. 실시예 2의 방법에서 여과필터 공극의 크기를 0.45um와 0.22um로 실시하고, 각각의 여과 전후의 세포 유래 베시클 입자수, 단백질 수, 크기, PDI(polydispersity index, 다분산지수)를 비교하였으며, 그 결과를 도 7에 나타내었다.The size of the pore size of the filtration filter showing an excellent purification effect in cell-derived vesicle purification and separation was selected. In the method of Example 2, the size of the filtration filter pore size was 0.45um and 0.22um, and the number of cell-derived vesicle particles, the number of proteins, the size, and PDI (polydispersity index) were compared before and after each filtration. , the results are shown in FIG. 7 .
도 7에 나타낸 바와 같이, 여과필터의 공극 크기가 0.45um인 경우 세포 유래 베시클의 입자 손실률이 현저히 적으며, 세포 유래 베시클의 크기가 100 내지 200nm이며, 세포 유래 베시클 PDI가 0.2 내지 0.3에 해당하여, 세포 유래 베시클의 균일성이 유지되는 것을 확인하였는바, 여과필터 공극의 크기를 0.45um로 선정하였다. 7, when the pore size of the filtration filter is 0.45um, the particle loss rate of the cell-derived vesicle is remarkably low, the size of the cell-derived vesicle is 100 to 200 nm, and the cell-derived vesicle PDI is 0.2 to 0.3 Corresponding to this, it was confirmed that the uniformity of the cell-derived vesicle was maintained, and the size of the pore size of the filtration filter was selected to be 0.45 μm.
실시예 4. 중간엽 줄기세포 유래 베시클의 분리 및 정제 방법에 따른 수율 및 순도 평가Example 4. Evaluation of yield and purity according to the method for isolation and purification of mesenchymal stem cell-derived vesicles
4.1 수율 평가4.1 Yield Assessment
실시예 2의 방법으로 중간엽 줄기세포 유래 베시클을 분리 및 정제한 결과, 4.44 X 10 4 ± 8.76 X 10 3 particle/cell 의 높은 수율로 세포 유래 베시클을 수득할 수 있음을 확인하였으며, 이를 그래프화하여 도 8에 나타내었다. 또한, 기존의 엑소좀, 엑소좀 유사체 또는 세포외소포를 정제하는 방법에 의한 수율을 표 1에 나타내었다.As a result of isolating and purifying mesenchymal stem cell-derived vesicles by the method of Example 2, it was confirmed that cell-derived vesicles can be obtained with a high yield of 4.44 X 10 4 ± 8.76 X 10 3 particle/cell, It is graphed and shown in FIG. 8 . In addition, the yield by the method of purifying the existing exosomes, exosome analogs or extracellular vesicles is shown in Table 1.
CategoryCategory Purification
methodPurification
method 		Patental cellPatent cell particles/ugparticles/ug ReferenceReference
EVsEVs TFFTFF hMSC
(inflammation-stimulated)hMSC
(inflammation-stimulated) 		3.3E+2/cell3.3E+2/cell Harting MT et.al. 2017Harting MT et.al. 2017
EVsEVs UCUC BM-MSCBM-MSC 4.3E+2/cell4.3E+2/cell Monica Reis et.al. 2018Monica Reis et. al. 2018
ExosomeExosome UCUC UC-MSC
(mechanical stimulated)UC-MSC
(mechanical stimulated) 		1.0E+4/cell1.0E+4/cell Litao Yan et.al. 2020Litao Yan et.al. 2020
ExosomeExosome UCUC BM-MSCBM-MSC 6.0E+2/cell6.0E+2/cell Senthikumar Kalimuthu et.al. 2018Senthikumar Kalimuthu et.al. 2018
Exosome
mimeticsExosome
mimetics 		UCUC BM-MSCBM-MSC 5.2E+3/cell5.2E+3/cell Senthikumar Kalimuthu et. al. 2018Senthikumar Kalimuthu et. al. 2018
표 1에 나타낸 기존 정제 방법의 수율과 비교하여, 본 발명의 정제 방법에 의하면 세포 유래 베시클을 4 내지 100 배 이상의 수율로 수득하는 것임을 확인하였다.Compared with the yield of the conventional purification method shown in Table 1, it was confirmed that the cell-derived vesicle was obtained in a yield of 4 to 100 times or more according to the purification method of the present invention.
4.2 순도(purity) 평가4.2 Purity evaluation
실시예 2의 방법으로 분리 및 정제된 중간엽 줄기세포 유래 베시클의 순도를 평가하기 위해, 세포 유래 베시클과 단백질의 양을 실시예 3.1과 동일한 방법으로 측정하고, 순도를 계산하였으며, 그 결과를 그래프화하여 도 9에 나타내었다.In order to evaluate the purity of the mesenchymal stem cell-derived vesicles isolated and purified by the method of Example 2, the amounts of the cell-derived vesicles and the protein were measured in the same manner as in Example 3.1, and the purity was calculated. was graphed and shown in FIG. 9 .
도 9에 나타낸 바와 같이, 실시예 2의 방법으로 세포 유래 베시클을 수득하는 경우, 3.86 X 10 9 ± 7.37 X 10 8 particle/ug 의 높은 순도로 세포 유래 베시클을 수득할 수 있음을 확인하였다. 또한, 기존의 엑소좀, 엑소좀 유사체 또는 세포외소포를 정제하는 방법에 의한 순도를 표 2에 나타내었다.As shown in FIG. 9 , when cell-derived vesicles were obtained by the method of Example 2, it was confirmed that cell-derived vesicles could be obtained with a high purity of 3.86 X 10 9 ± 7.37 X 10 8 particle/ug. . In addition, the purity by the method of purifying the existing exosomes, exosome analogues or extracellular vesicles is shown in Table 2.
CategoryCategory Purification
methodPurification
method 		Patental cellPatent cell particles/ugparticles/ug ReferenceReference
ExosomeExosome UCUC UC-MSC
(mechanical
stimulated)UC-MSC
(mechanical
stimulated) 		1.E+061.E+06 Litao Yan et.al. 2020Litao Yan et.al. 2020
ExosomeExosome TFFTFF BM MSCBM MSC 3.7E+8±2.1E+83.7E+8±2.1E+8 Alexandra Alvarez Fernandex et.al. 2018Alexandra Alvarez Fernandex et.al. 2018
ExosomeExosome TFFTFF ADSCADSC 1.37E+071.37E+07 조용우 et.al. 2017Cho et.al. 2017
ExosomeExosome UCUC BM-MSCBM-MSC 3.2E+083.2E+08 Senthikumar Kalimuthu et.al. 2018Senthikumar Kalimuthu et.al. 2018
Exosome
mimeticsExosome
mimetics 		UCUC BM-MSCBM-MSC 1.0E+091.0E+09 Senthikumar Kalimuthu et.al. 2018Senthikumar Kalimuthu et.al. 2018
표 2에 나타낸 기존 정제 방법의 순도와 비교하여, 본 발명의 정제 방법에 의하면 세포 유래 베시클을 약 4 내지 1000 배 이상의 순도로 수득하는 것임을 확인하였다.Compared with the purity of the conventional purification method shown in Table 2, it was confirmed that the cell-derived vesicle was obtained with a purity of about 4 to 1000 times or more according to the purification method of the present invention.
4.3. 다른 정제 방법과 비교 4.3. Comparison with other purification methods
본 발명의 실시예 2의 정제방법을 이용하면, 세포 유래 베시클을 다른 정제 방법 대비 더욱 우수한 수득률, 높은 순도로 정제할 수 있는지 구체적인 비교 실험을 통해 확인하였다. 중간엽 줄기세포 유래 베시클을 정제 수득하기 위하여 종래 알려진 엑소좀 분리 방법인 농도구배초원심분리(gradient ultracentrifugation, gUC), 초원심분리(ultracentrifugation, UC) 방법을 수행하고, 본 발명의 실시예 2와 입자 수득률 및 순도를 비교하였다. gUC 방법은 Crude CDV를 50 및 10% Opti-prep 용액에 적층하고 120,000g, 4℃에서 2hr 동안 초원심분리하여 수행하였다. UC 방법은 Crude CDV를 3,000g, 4℃에서 10 min 동안 원심분리한 후, 상층액을 순차적으로 10,000g, 4℃에서 30 min 및 120,000g, 4℃에서 2hr 동안 초원심분리하여 수행하였다. 수득된 줄기세포 유래 베시클의 입자 수득률과 순도 비교 결과를 도 10에 나타내었다. Using the purification method of Example 2 of the present invention, it was confirmed through a specific comparative experiment whether the cell-derived vesicles could be purified with better yield and higher purity than other purification methods. In order to purify and obtain mesenchymal stem cell-derived vesicles, conventional known exosome separation methods such as gradient ultracentrifugation (gUC) and ultracentrifugation (UC) were performed, and Example 2 of the present invention and particle yield and purity were compared. The gUC method was performed by stacking Crude CDV on 50 and 10% Opti-prep solutions and ultracentrifugation at 120,000 g, 4° C. for 2 hr. The UC method was performed by centrifuging Crude CDV at 3,000 g, 4° C. for 10 min, and then ultracentrifuging the supernatant sequentially at 10,000 g, 30 min and 120,000 g at 4° C., and 4° C. for 2 hr. The results of comparison of particle yield and purity of the obtained stem cell-derived vesicles are shown in FIG. 10 .
도 10에 나타낸 바와 같이, 본 발명의 정제방법(TFF으로 표기) 을 이용하면 다른 기존의 엑소좀 분리 방법 대비 약 1.5 배 내지 2 배의 입자 수득률 증가가 나타났으며, 순도는 약 1.3 배 내지 3 배 증가됨을 확인하였다. As shown in FIG. 10, when the purification method of the present invention (denoted as TFF) was used, the particle yield increased by about 1.5 to 2 times compared to other existing exosome separation methods, and the purity was about 1.3 to 3 times. It was confirmed that the fold increased.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다.Above, specific parts of the present invention have been described in detail, for those of ordinary skill in the art, these specific descriptions are only preferred embodiments, and it is clear that the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (15)

  1. a) 세포 유래 베시클을 포함하는 시료를 60 내지 120 ml/분의 유량(flow rate) 및 0.1 내지 0.3 bar의 막간차압(transmembrane pressure)을 유지하며, 분자량 차단(molecular weight cutoff, MWCO)이 500 내지 1,000kDa인 십자류여과(tangential flow filtration, TFF) 필터를 사용하여, 십자류여과를 실시하는 단계; 및 a) maintaining a flow rate of 60 to 120 ml/min and a transmembrane pressure of 0.1 to 0.3 bar for a sample containing a cell-derived vesicle, and a molecular weight cutoff (MWCO) of 500 to 1,000 kDa using a tangential flow filtration (TFF) filter, performing cross flow filtration; and
    b) 공극의 크기가 0.2 내지 0.5μm인 필터를 이용하여 여과하는 단계를 포함하고, b) filtration using a filter having a pore size of 0.2 to 0.5 μm,
    상기 a) 단계의 십자류여과는 The cross flow filtration of step a)
    1) 상기 시료의 초기 부피의 20 내지 40 %가 되도록 한외여과를 통해 농축하는 단계;1) Concentrating through ultrafiltration to 20 to 40% of the initial volume of the sample;
    2) 농축이 끝난 부피 기준 4 내지 8배의 부피를 갖는 완충용액을 이용하여 탈염과 버퍼교환을 수행하는 단계; 및2) performing desalting and buffer exchange using a buffer solution having a volume of 4 to 8 times the volume of the concentrated volume; and
    3) 상기 시료의 초기 부피의 3 내지 7 %가 되도록 한외여과를 통해 재농축하는 단계를 포함하는 것인, 세포 유래 베시클의 분리 및 정제 방법.3) The method of separating and purifying cell-derived vesicles, comprising the step of re-concentrating through ultrafiltration to 3 to 7% of the initial volume of the sample.
  2. 제1항에 있어서, 상기 세포 유래 베시클을 포함하는 시료는 세포를 포함하는 시료를 미세공극에 압출하는 단계를 포함하는 제조방법에 의해 제조되는 것인, 세포 유래 베시클의 분리 및 정제 방법.The method of claim 1, wherein the sample containing the cell-derived vesicle is prepared by a manufacturing method comprising the step of extruding the sample containing the cells into micropores.
  3. 제2항에 있어서, 상기 미세공극에 압출하는 단계는 미세공극의 크기가 큰 것으로부터 미세공극의 크기가 작은 것으로 순차적으로 압출하는 단계인, 세포 유래 베시클의 분리 및 정제 방법.The method of claim 2, wherein the extruding into the micropores is a step of sequentially extruding from a micropore having a large size to a micropore having a smaller size.
  4. 제2항에 있어서, 상기 세포는 줄기세포, 선포세포, 근상피세포, 적혈구, 단핵구, 수지상 세포, 자연살해 세포 및 혈소판으로 이루어진 군에서 선택된 어느 하나 이상인, 세포 유래 베시클의 분리 및 정제 방법.The method of claim 2, wherein the cells are at least one selected from the group consisting of stem cells, acinar cells, myoepithelial cells, red blood cells, monocytes, dendritic cells, natural killer cells, and platelets.
  5. 제4항에 있어서, 상기 줄기세포는 중간엽 줄기세포, 유도만능줄기세포, 배아줄기세포 및 침샘 줄기세포로 이루어진 군에서 선택된 어느 하나 이상인, 세포 유래 베시클의 분리 및 정제 방법. The method of claim 4, wherein the stem cells are at least one selected from the group consisting of mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells and salivary gland stem cells.
  6. 제1항에 있어서, 상기 탈염과 버퍼교환은 연속적으로 수행되는 것인, 세포 유래 베시클의 분리 및 정제 방법.The method of claim 1, wherein the desalting and buffer exchange are continuously performed.
  7. 제1항에 있어서, 상기 막간차압(transmembrane pressure)은 0.2 bar인, 세포 유래 베시클의 분리 및 정제 방법.The method of claim 1, wherein the transmembrane pressure is 0.2 bar.
  8. 제1항에 있어서, 상기 분자량 차단(molecular weight cutoff, MWCO)은 700 내지 800kDa인, 세포 유래 베시클의 분리 및 정제 방법.The method of claim 1, wherein the molecular weight cutoff (MWCO) is 700 to 800 kDa.
  9. 제1항에 있어서, 상기 2) 단계는 농축이 끝난 부피 기준 5 내지 7 배의 부피를 갖는 완충용액을 이용하여 탈염과 버퍼교환을 수행하는 단계이고, 상기 3) 단계는 상기 시료의 초기 부피의 3 내지 7%이 되도록 한외여과를 통해 재농축하는 단계인, 세포 유래 베시클의 분리 및 정제 방법.The method according to claim 1, wherein step 2) is a step of performing desalting and buffer exchange using a buffer solution having a volume 5 to 7 times the volume based on the finished concentration, and step 3) is the initial volume of the sample. A method of separating and purifying cell-derived vesicles, which is a step of re-concentration through ultrafiltration to 3 to 7%.
  10. 제1항에 있어서, 상기 세포 유래 베시클의 분리 및 정제 방법은 십자류 여과를 실시하는 단계 이전에 세포 유래 베시클을 포함하는 시료에 핵산분해효소, 40 내지 60 mM Tris-HCl 및 1 내지 3 mM 염화마그네슘을 시료 내 DNA량에 비례하여 처리하는 단계를 더 포함하는 것인, 세포 유래 베시클의 분리 및 정제 방법.The method according to claim 1, wherein, in the method for separating and purifying cell-derived vesicles, nuclease, 40 to 60 mM Tris-HCl, and 1 to 3 The method of separating and purifying cell-derived vesicles further comprising the step of treating mM magnesium chloride in proportion to the amount of DNA in the sample.
  11. 제1항에 있어서, 상기 세포 유래 베시클의 분리 및 정제 방법은 상기 1) 단계 직전 및 상기 b) 단계 직전에 20 내지 25℃인 실온에서 10분간 2000 내지 4000 x G 조건으로 원심분리하는 단계를 더 포함하는 것인, 세포 유래 베시클의 분리 및 정제 방법.The method of claim 1, wherein the method for separating and purifying cell-derived vesicles comprises the steps of centrifugation at 2000 to 4000 x G conditions for 10 minutes at 20 to 25° C. just before step 1) and immediately before step b). The method of isolation and purification of cell-derived vesicles further comprising.
  12. 제1항에 있어서, 상기 b) 단계의 필터는 공극의 크기가 0.3 내지 0.5 μm인, 세포 유래 베시클의 분리 및 정제 방법.The method of claim 1, wherein the filter in step b) has a pore size of 0.3 to 0.5 μm.
  13. 제1항에 있어서, 상기 분리 및 정제된 세포 유래 베시클은 크기가 100 내지 200nm이며, 다분산 지수(polydispersity index, PDI)가 0.2 내지 0.5인, 세포 유래 베시클의 분리 및 정제 방법.The method of claim 1, wherein the isolated and purified cell-derived vesicle has a size of 100 to 200 nm and a polydispersity index (PDI) of 0.2 to 0.5.
  14. 제1항 내지 제13항 중 어느 한 항의 세포 유래 베시클의 분리 및 정제 방법에 의해 분리 및 정제된 세포 유래 베시클.A cell-derived vesicle isolated and purified by the method for separating and purifying a cell-derived vesicle according to any one of claims 1 to 13.
  15. 제14항에 있어서, 상기 세포 유래 베시클은 크기가 100 내지 200nm이며, 다분산 지수(polydispersity index, PDI)가 0.2 내지 0.5인, 세포 유래 베시클.The cell-derived vesicle of claim 14 , wherein the cell-derived vesicle has a size of 100 to 200 nm and a polydispersity index (PDI) of 0.2 to 0.5.
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