WO2021221368A1 - Procédé de filtration de vésicule dérivée de cellules - Google Patents

Procédé de filtration de vésicule dérivée de cellules 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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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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

La présente invention concerne un procédé pour isoler des vésicules dérivées de cellules à une pureté élevée et filtrer sélectivement des vésicules dérivées de cellules à partir d'une suspension contenant des vésicules dérivées de cellules et, plus spécifiquement, un procédé pour isoler et filtrer sélectivement des vésicules dérivées de cellules à partir d'une suspension contenant des vésicules dérivées de cellules par une discrimination efficace contre les impuretés : débris cellulaires, déchets, protéines et macroparticules. En utilisant le procédé d'isolement et de filtration de vésicules dérivées de cellules selon la présente invention, des vésicules dérivées de cellules peuvent être isolées et filtrées avec un rendement élevé et une pureté élevée.
PCT/KR2021/004883 2020-04-28 2021-04-19 Procédé de filtration de vésicule dérivée de cellules WO2021221368A1 (fr)

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KR20200051513 2020-04-28
KR10-2020-0051513 2020-04-28
KR1020200180443A KR20210133116A (ko) 2020-04-28 2020-12-22 세포 유래 베시클의 정제 방법
KR10-2020-0180443 2020-12-22

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WO2024002311A1 (fr) * 2022-06-29 2024-01-04 Beijing Theraxyte Bioscience Co. Ltd. Procédés de fabrication et d'utilisation de vésicules extracellulaires

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