WO2020004878A1 - Size-based separation method for highly concentrating extracellular vesicle from fluid sample - Google Patents

Size-based separation method for highly concentrating extracellular vesicle from fluid sample Download PDF

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WO2020004878A1
WO2020004878A1 PCT/KR2019/007593 KR2019007593W WO2020004878A1 WO 2020004878 A1 WO2020004878 A1 WO 2020004878A1 KR 2019007593 W KR2019007593 W KR 2019007593W WO 2020004878 A1 WO2020004878 A1 WO 2020004878A1
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extracellular vesicles
filter
fluid sample
concentration
pipette tip
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PCT/KR2019/007593
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French (fr)
Korean (ko)
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김준호
유가은
이정선
장송선
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주식회사 바이오솔루션
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Priority claimed from KR1020180171163A external-priority patent/KR102170903B1/en
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Priority to US17/253,388 priority Critical patent/US20210262908A1/en
Publication of WO2020004878A1 publication Critical patent/WO2020004878A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples

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  • the present invention is a method for concentrating extracellular vesicles from a fluid sample, specifically using a filter having an average pore size of 20 to 100 nm, using a fluid sample containing a surfactant and an elution buffer containing a gas
  • a new method of eluting extracellular vesicles bound to a filter relates to a method for concentrating extracellular vesicles with high efficiency in a short time.
  • Extracellular vesicles are classified into microvesicles and exosomes, and serve as mutual information exchange between cells, and function of biomarkers in connection with cancer cell metastasis, immunity, and tissue regeneration. Do it.
  • a separator for separating extracellular vesicles circulating in blood vessels from blood has been developed, such as a centrifugal separator.
  • the centrifugal separator captures the endoplasmic reticulum from nanosized materials formed into pellets using centrifugal force.
  • the sedimentation rate of the particles is very low, and as a result, centrifugation of these particles takes several minutes to several hours, resulting in a lot of manpower and time required to unload the pellet.
  • the endoplasmic reticulum can aggregate and cause a precipitate.
  • the centrifugation method when using the centrifugation method, not only takes a long time, but also can separate only about 5% to 25% of the total amount of extracellular vesicles contained in the fluid, except for most of the cells (75% to 95%) Extra vesicles are lost.
  • the centrifugation method that requires an ultra-high speed centrifugal separator has not only a high cost aspect of the equipment, but also a limitation that the amount of fluid allowed at one time is up to 0.6 L (Sci Rep. (2015) Aug 14; 5: 13103).
  • the present inventors studied to develop a method for concentrating extracellular vesicles with high efficiency in a short time, and concentrated a large amount of extracellular vesicles in a short time by controlling the use of specific pore size filter and fluid sample composition and elution buffer.
  • the present invention has been completed by discovering that the loss of extracellular vesicles can be reduced.
  • An object of the present invention is a method of concentrating extracellular vesicles from a fluid sample with high efficiency within a short time, specifically using a filter having an average pore size of 20 to 100 nm and adjusting the use of the fluid sample composition and the elution buffer to extracellularly.
  • a method for concentrating endoplasmic reticulum with high efficiency is a method of concentrating extracellular vesicles from a fluid sample with high efficiency within a short time, specifically using a filter having an average pore size of 20 to 100 nm and adjusting the use of the fluid sample composition and the elution buffer to extracellularly.
  • the extracellular vesicle enrichment method of the present invention When the extracellular vesicle enrichment method of the present invention is used, the extracellular vesicles can be concentrated with high efficiency while reducing the concentration time while simplifying the concentration process. This suggests an economical enrichment method suitable for extracellular vesicles by reducing time and cost and increasing enrichment efficiency compared to conventional enrichment methods.
  • the properties of the filters, fluid samples and elution buffers used in the present invention are useful in that they can be applied to a variety of known equipment without being limited to specific equipment.
  • FIG. 1 shows a concentrated pipette tip for use in the present invention.
  • Figure 2 shows the total volume of culture separated from the extracellular vesicles in a manner using a 50 nm filter.
  • Figure 3 shows the total volume of the culture separated from the extracellular vesicles in a manner using an ultrafilter ( ⁇ 20 nm).
  • Figure 4 shows the number of extracellular vesicle particles concentrated per unit volume according to the type of filter. Fold is a value confirming the concentration ratio compared to the culture medium (culture medium).
  • Figure 5 shows the total particle number of the extracellular vesicles obtained according to the type of filter.
  • Figure 6 shows the relative impurity content according to the type of elution buffer.
  • FIG. 9 shows the filter efficiency without concentration and the efficiency of obtaining extracellular vesicles (Concentration EVs) according to filter size ( ⁇ 20 nm, 50 nm, 100 nm and 200 nm) with fixed use count of elution buffer (3 times). It is a graph showing the removal efficiency of extracellular vesicles (Filtrared EVs) discharged through.
  • 10 is a graph comparing the yield efficiency of the extracellular vesicles concentrated by the concentration method of the present invention and the concentration method using ExoQuick.
  • 11 is an image confirming the appearance of the extracellular vesicles concentrated by the concentration method of the present invention and the concentration method using ExoQuick.
  • FIG. 12 is a diagram comparing the size of the extracellular vesicles concentrated by the concentration method of the present invention and the concentration method using ExoQuick.
  • Figure 13 is a diagram showing the cost required to concentrate the extracellular vesicles by the concentration method and ExoQuick concentration method of the present invention.
  • FIG. 14 is a graph showing the efficiency of obtaining extracellular vesicles according to the inclusion concentration (0%, 0.05%, 1%, 5% and 10%) of surfactant in the fluid sample.
  • a first aspect of the invention is a method of concentrating extracellular vesicles from a fluid sample
  • a concentrated pipette tip comprising a filter, an opening, and an outlet having an average pore size of 20 to 100 nm
  • a concentrating unit configured to aspirate a fluid sample through the condensation pipette tip and to recover the condensed extracellular vesicles from the condensation pipette tip;
  • an extracellular vesicle separation method that is frequently used is a method using a filter.
  • Filtering is a method of filtering extracellular vesicles using a filter having a pore size or similar to that of extracellular vesicles.
  • Extracellular vesicles are filtered out and other proteins are discharged so that the rate of separation can be controlled by the speed of the fluid, and the separation time is fast because no reaction time is required.
  • the filter size and the constituents of the elution buffer it is characterized by lowering the ratio of extracellular vesicles bound to or out of the filter to increase the final separation efficiency.
  • the present inventors studied to develop a method for concentrating extracellular vesicles in a short time at low cost, (i) using a filter having an average pore size of 20 to 100 nm, (ii) Tris elution buffer Repeatedly eluting with (Tris elution buffer) or PBS® elution buffer, an elution buffer containing a gas for foam formation, and (iii) adding an additional surfactant to the fluid sample containing extracellular vesicles.
  • Tris elution buffer Repeatedly eluting with (Tris elution buffer) or PBS® elution buffer, an elution buffer containing a gas for foam formation
  • adding an additional surfactant to the fluid sample containing extracellular vesicles.
  • the present invention is a method for concentrating extracellular vesicles using a filter and an elution buffer.
  • the concentrating device used in the present invention comprises (i) a container containing a fluid sample; (ii) a concentrated pipette tip comprising a filter, an opening, and an outlet having an average pore size of 20 to 100 nm; And (iii) a concentrating unit configured to aspirate a fluid sample through the condensation pipette tip and to recover the condensed extracellular vesicles from the condensation pipette tip.
  • the condensation pipette tip may be a separate elution port. That is, although the opening included in the concentrated pipette tip may simultaneously perform suction and elution of the sample, when the concentration pipette tip further includes an elution port, the fluid sample and the eluent may be sucked through different portions. .
  • FIG. 1 (a) shows the concentrated pipette tip 100. Specifically, a thickening pipette tip 100 comprising an opening 105 and a filter 101 is shown. Concentrated pipette tip 100 also includes a filter 101, a permeate purge 107, and a potting 103 of permeate draw 109. The connection 113 shown in the enrichment pipette tip 100 allows the enrichment pipette tip 100 to be connected to a enrichment unit for operation of the enrichment pipette tip 100.
  • Figure 1 (b) shows a connection portion 113 including three ports.
  • the connection part 11 includes a first port 115 connected to the permeate purge 107, a second port 117 connected to the filter 101, and a third port 119 connected to the permeate drawer 109. There is.
  • the first port 115, the second port 117, and the third port 119 are connected to the concentration unit through the connection portion 113, and the concentration pipette tip 100 is connected to the concentration unit.
  • the fluid sample is sucked into the lower opening 105 and passes through the porous surface of the filter 101 using a pump connected to the permeate draw 109 through the third port 119.
  • the filter 101 or other thin film filter is a dry hydrophilic filter, a hydrophilic filter filled with glycerin, or another type of filter that allows air to pass through at first, and passes the liquid upon contact with the liquid. That is, air is sucked into the lower opening 105 and the fluid sample passes through the porous surface of the filter 101 until it is sucked into the lower opening 105 and contacts the filter 101 and passes through the porous surface.
  • the concentration system used in the present invention is as follows. All of the tubes to which the disposable thickening pipette tip 100 couples to the thickening unit are connected at a single connection point located at the top of the thickening pipette tip 100.
  • the concentration pipette tip 100 functions with the system including the concentration unit and the fluid sample.
  • the concentrating unit operates by immersing the opening 105 of the condensing pipette tip 100 in a fluid sample contained in a suitable sample container. The fluid sample is then sucked into the concentrated pipette tip 100 and brought into contact with the filter 101. The liquid passes through the filter 101, while particles similar to or larger than the pore size of the filter 101 are collected and retained in the filter 101.
  • the fluid is removed to leave only the collected sample, by dipping the lower opening 105 of the concentrated pipette tip 100 into a suitable container and eluting the collected material using an eluent, Obtained concentrated extracellular vesicles.
  • the method for concentrating the extracellular vesicles of the present invention comprises aspirating a fluid sample through the concentrated pipette tip and eluting the extracellular vesicles collected in the filter of the concentrated pipette tip.
  • the fluid sample may further comprise 0.05 to 5% by weight of surfactant.
  • the surfactant may include 0.1 to 4% by weight, 0.3 to 3% by weight, 0.5 to 2% by weight, 0.75 to 1.5% by weight, more specifically 1% by weight.
  • the surfactant of the present invention may be poloxamer, polysorbate, or a combination thereof.
  • the surfactant may be poloxamer 188, polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 80 (Tween 80) and / or their It is characterized in that it is selected from the group consisting of a combination. In the embodiment of the present invention it was carried out using Tween 20 as a representative surfactant.
  • the elution may be performed using an elution buffer comprising a Tris elution buffer or a PBS elution buffer.
  • the number of elution may be 1 to 5 times.
  • the elution buffer may further include a bubble forming gas, more specifically carbon dioxide, nitrogen, argon, air, liquefied petroleum gas, or a combination thereof.
  • a bubble forming gas more specifically carbon dioxide, nitrogen, argon, air, liquefied petroleum gas, or a combination thereof.
  • the average pore size of the filter 101 used for collecting extracellular vesicles may be 20 to 100 nm, more specifically 30 to 80 nm, 40 to 60 nm, or 50 nm. This is an appropriate size to more effectively capture extracellular vesicles.
  • the size of the extracellular vesicles is known from 20 nm to 1 um.
  • a filter having an average pore size of less than 20 nm is used, other liquids or proteins except the extracellular vesicles may have a long time to pass through the filter or the filter may be blocked, thereby preventing the extracellular vesicles from being concentrated.
  • the average pore size of the filter is larger than the extracellular vesicles, there is a problem that the rate at which the extracellular vesicles are lost increases.
  • the average pore size of the filter is more than 100 nm, the proportion of small extracellular vesicles (exosomes) having a size of about 20 to 100 nm in the extracellular vesicles is a problem.
  • a filter having an optimal pore size should be used.
  • it is characterized by selecting a filter having an average pore size of 20 to 100 nm as an efficient filter that can increase the total amount of extracellular vesicles recovered.
  • the present invention determines the conditions of the average pore size of the filter having an average pore size suitable for concentrating the extracellular vesicles, the composition change of the fluid sample and the composition of the elution buffer, the number of elution, etc. I found a combination.
  • the surface area of the filter of the present invention may be 5 cm 2 to 20 m 2 , but is not limited thereto. Any filter having an average pore size of 20 to 100 nm can be applied to the concentration method of the present invention regardless of the surface area.
  • a tris elution buffer or PBS elution buffer can be used as a solution for eluting the extracellular vesicles collected in the filter 101.
  • a tris elution buffer or PBS elution buffer can be used as a solution for eluting the extracellular vesicles collected in the filter 101.
  • PBS elution buffer In order to determine the concentration of small impurities (nanoparticle) inside the eluate, it is important to reduce the amount of mixing in the extracellular vesicles to be concentrated afterwards to a minimum.
  • the number of eluents used is important to increase the elution efficiency of the extracellular vesicles.
  • the number of eluents used that is, the number of elutions is 1 to 5 times. If the number of elution is more than five times, the amount of extracellular vesicles to be separated may increase, but there may be a problem of diluting the concentration of the extracellular vesicles or breaking the shape.
  • Extracellular vesicles (Extracellular vesicles) of the present invention is a endoplasmic reticulum produced in the cells and secreted out of the cell, proteins of various functions released from the cell to the extracellular environment, such as various growth factors (Growth factors), chemokines ( Various biomolecules such as chemokines, cytokines, transcription factors, RNAs (mRNA, miRNA, etc.), lipids, etc. Means. Exosomes, microvesicles, microparticles, and the like, but are not limited thereto.
  • Enriched extracellular vesicles can be applied to assay methods commonly used in the art. For example, immunoassay, polymerase chain reaction (PCR), electrochemical, microarray, flow cytometry, biosensor, lab-on-chip a-chip) and rapid growth based detection, but are not limited thereto.
  • assay methods commonly used in the art. For example, immunoassay, polymerase chain reaction (PCR), electrochemical, microarray, flow cytometry, biosensor, lab-on-chip a-chip) and rapid growth based detection, but are not limited thereto.
  • the main technical features in the concentration method of the present invention is the type of filter, the type of elution buffer, and the number of times of use.
  • Embodiments of various concentration methods using filters available in the art are all within the scope of the present invention.
  • the method of the present invention may further comprise the step of separating the extracellular vesicles.
  • the extracellular vesicles eluted by the method of the present invention may be used in addition to, but are not limited to, various known extracellular vesicle separation methods.
  • the extracellular vesicle separation method may be, but is not limited to, polymer-based separation, centrifugation-based separation, concentration-based separation, and filtration-based separation.
  • the fat stem cells were cultured in a culture dish at about 80-90% and then replaced with MEM-alpha (Gibco) medium containing 10% of serum replacement (Serum replacement, Gibco). Two days later, the cultured cells of the adipose stem cells were collected in a tube, the dead cells were allowed to settle by centrifuge (3,000 g, 20 minutes), and the supernatant was separated. The obtained supernatant was used in the following examples, hereinafter referred to as 'culture medium'.
  • the filter of 50 nm pore size When the filter of 50 nm pore size was used, the total volume of the culture liquid passing through the filter was steadily increased as the filter was less clogged and the concentration was increased to 55 ml / 6.5 minutes. In addition, it was finally confirmed that the total amount of the culture medium is about 60 ml (Fig. 2). On the other hand, when the ultrafilter was used, the maximum concentration of the culture medium was only about 30 ml, and the concentration of the concentrate did not increase over a certain level even after time, and finally, the concentration was reached at an average rate of 27 ml / 6.5 minutes. Was calculated (FIG. 3).
  • Liquid substances are likely to contain impurities of similar size to extracellular vesicles. Therefore, the presence of impurities in the elution buffer was tested for confirmation. Relative amounts and sizes of impurities in each elution buffer were measured by Nanoparticle tracking analysis equipment (Malvern, LM10). Buffers 1 and 3 are PBS elution buffers and Buffers 2 and 4 are Tris elution buffers.
  • the impurities have a range of sizes ranging from 20 nm to 1 um, which is nanoscale, and thus, it is highly difficult to distinguish them from extracellular vesicles. Therefore, the test was performed by selecting an elution buffer containing few impurities.
  • the recovery rate shows the ratio of the extracellular vesicles separated from the total extracellular vesicles contained in the culture medium.
  • the recovery was 60% in the group using the elution buffer once, 90% recovery in the group used three times. In other words, the recovery rate was increased by increasing the number of elutions to three times.
  • Ultrafilters ( ⁇ 20 nm), 50 nm filters, 100 nm filters and 200 nm filters were used to compare the total yield according to the size of the filter.
  • the 50 nm filter showed a high recovery (black graph, Concentrated EVs) close to about 90%, while the ultra filter, 100 nm filter and 200 nm filter had a low recovery of about 50%. Indicated.
  • 10 ml of the culture solution was aspirated into a 50 nm filter and eluted using Tris elution buffer three times.
  • 10 ml of the culture was mixed with 2 ml of ExoQuick, and then waited for the extracellular vesicles to elute from the culture for 24 hours.
  • Eluted extracellular vesicles were separated by sinking in a centrifuge (1500 g) for 30 minutes. Each isolated extracellular vesicles were analyzed by Nanoparticle tracking analysis equipment (Malvern, LM10).
  • the extracellular vesicle images measured by Nanosight showed that the extracellular vesicles separated according to the enrichment method of the present invention are identical to the extracellular vesicles separated using the culture medium and Exoquick (Fig. 11).
  • the size of the extracellular vesicles separated according to the concentration method of the present invention and the size of the extracellular vesicles separated using the culture medium and Exoquick was found to be similar (Fig. 12).
  • ExoQuick is a package that requires an ExoQuick kit and centrifuge equipment. Concentrated pipettes require filters and elution buffers as consumables and pumps as equipment. As shown in Figure 13, when compared to the cost of consumables, the cost of concentrating extracellular vesicles in a 60 ml culture solution was confirmed to be about 600,000 won for Exoquick and about 40,000 won for the concentrated pipette. When using the concentrated pipette of the present invention compared to ExoQuick was confirmed that the cost can be reduced by at least 15 times.
  • Tween 20 was made into 0%, 0.05%, 1%, 5%, and 10% in 10 ml of culture. Each culture solution was inhaled into a 50 nm filter, and the extracellular vesicles bound to the filter were eluted using Tris elution buffer three times. The extracellular vesicles were measured using a nanoparticle tracking analysis device (Malvern, LM10) to compare the recovery rates.
  • a nanoparticle tracking analysis device Malvern, LM10
  • the separation method by adding a surfactant to the fluid sample can increase the separation efficiency of the extracellular vesicles, but the appropriate concentration was found to be about 1%.

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Abstract

The present invention relates to a method for concentrating extracellular vesicles from a fluid sample and, more particularly, to a method for concentrating extracellular vesicles within a short time at a high yield by using a 20-100 nm filter and controlling the composition of a fluid sample and the use of an eluting buffer. The use of the extracellular vesicle concentrating method of the present invention can simplify a concentration process, reduce a concentration time, and concentrate extracellular vesicles at high yield. The method reduces time and cost and increases a concentration yield, compared to conventional concentration methods and is proposed as an economically advantageous concentration method suitable for extracellular vesicles.

Description

유체 샘플로부터 세포 외 소포체를 고농축하는 크기 기반 분리법Size-based Separation of High Concentration of Extracellular Vesicles from Fluid Samples
본 발명은 유체 샘플로부터 세포 외 소포체(Extracellular vesicle)를 농축하는 방법으로서, 구체적으로 평균 기공 크기가 20 내지 100 nm인 필터를 사용하고, 계면활성제가 포함된 유체 샘플과 가스가 포함된 용리 완충액으로 필터에 결합된 세포 외 소포체를 용리하는 새로운 방식으로 단시간 내에 높은 효율로 세포 외 소포체를 농축하는 방법에 관한 것이다.The present invention is a method for concentrating extracellular vesicles from a fluid sample, specifically using a filter having an average pore size of 20 to 100 nm, using a fluid sample containing a surfactant and an elution buffer containing a gas A new method of eluting extracellular vesicles bound to a filter relates to a method for concentrating extracellular vesicles with high efficiency in a short time.
현재 불치/난치 질환을 치료하기 위한 새로운 패러다임으로 성체줄기세포 치료법이 다양하게 임상 적용되고 있다. 그러나, 환자 혹은 공여자로부터 추출 과정, 선별된 줄기세포를 증식시키기 위해 체외 배양하는 과정 중 발생할 수 있는 이종 혈청(Xenogenic serum)의 인터널리제이션에 의한 인수전염(Zoonosis)의 문제, 줄기세포가 체내 이식되었을 때 왕성한 증식력과 상대적으로 큰 세포 사이즈 등의 줄기세포 특성으로 인해 발생할 수 있는 종양 형성(Tumor formation) 문제, 혈관 폐쇄 유발 경색(Vascular occlusion causing infarcts) 등의 위험 요소가 존재한다.As a new paradigm for treating incurable and incurable diseases, adult stem cell therapy has been applied to various clinical applications. However, problems of zoonoses caused by internalization of Xenogenic serum, which may occur during extraction from patients or donors, and in vitro culture to propagate selected stem cells, stem cells transplanted into the body There are risk factors such as tumor formation problems and vascular occlusion causing infarcts that can be caused by stem cell characteristics such as vigorous proliferation and relatively large cell size.
따라서, 앞서 언급한 살아있는 줄기세포를 직접 이용한 치료에서 발생할 수 있는 다양한 위험 요소 혹은 문제들을 회피하는 방안이 활발히 연구되고 있으며, 그 일환으로 줄기세포로부터 유래된 세포 외 소포체로 줄기세포 치료 기능을 대신할 수 있다는 연구 결과들이 나오고 있다(Cell. Mol. Life Sci. (2011)68: 2667-2688).Therefore, there are active researches on avoiding various risk factors or problems that may occur in the aforementioned treatments using living stem cells. As part of this, extracellular vesicles derived from stem cells can replace stem cell treatment functions. Research has been published (Cell. Mol. Life Sci. (2011) 68: 2667-2688).
세포 외 소포체는 미세소포체(Microvesicle)와 엑소좀(Exosome)등으로 구분되며, 세포간의 상호 정보교환의 역할을 하고 암세포의 전이, 면역, 조직의 재생 등과 연관되어 바이오마커(Bio-maker)의 기능을 한다.Extracellular vesicles are classified into microvesicles and exosomes, and serve as mutual information exchange between cells, and function of biomarkers in connection with cancer cell metastasis, immunity, and tissue regeneration. Do it.
특히, 혈관 내에서 순환하는 세포 외 소포체를 혈액과 분리하는 분리장치가 개발되었는데 원심분리장치를 그 예로 들 수 있다. 원심분리장치는 원심력을 이용하여 펠렛으로 형성된 나노사이즈의 물질들에서 소포체를 포집한다. 그러나, 미크론 크기 범위에서 입자의 침강 속도는 매우 낮으므로 결과적으로 이들 입자의 원심 분리는 수 분에서 수 시간이 걸리게 되며, 이로 인한 문제로 펠렛을 풀어내는 과정에서 많은 인력과 시간이 필요하다는 점과 소포체가 응집되어 침전물이 발생할 수 있다는 점이 있다. In particular, a separator for separating extracellular vesicles circulating in blood vessels from blood has been developed, such as a centrifugal separator. The centrifugal separator captures the endoplasmic reticulum from nanosized materials formed into pellets using centrifugal force. However, in the micron size range, the sedimentation rate of the particles is very low, and as a result, centrifugation of these particles takes several minutes to several hours, resulting in a lot of manpower and time required to unload the pellet. There is a point in which the endoplasmic reticulum can aggregate and cause a precipitate.
또한, 원심분리방법을 이용할 경우, 많은 시간이 소요될 뿐만 아니라, 유체에 포함된 세포 외 소포체 전체량 중 약 5%~25%만을 분리할 수 있고, 이를 제외한 대부분(75%~95%)의 세포 외 소포체를 잃어버리게 된다. 이와 함께 초고속 원심분리장치가 필요한 원심분리 방법은 장비의 고비용 측면뿐만 아니라, 한 회에 허용되는 유체의 양이 현재까지 최대 0.6 L 수준이라는 한계가 있다(Sci Rep. (2015) Aug 14;5:13103).In addition, when using the centrifugation method, not only takes a long time, but also can separate only about 5% to 25% of the total amount of extracellular vesicles contained in the fluid, except for most of the cells (75% to 95%) Extra vesicles are lost. In addition, the centrifugation method that requires an ultra-high speed centrifugal separator has not only a high cost aspect of the equipment, but also a limitation that the amount of fluid allowed at one time is up to 0.6 L (Sci Rep. (2015) Aug 14; 5: 13103).
본 발명자들은 단 시간 내에 높은 효율로 세포 외 소포체를 농축하는 방법을 개발하고자 연구하였고, 특정 기공 크기 필터 및 유체 샘플 조성 변화 및 용리 완충액의 사용 조절을 통해 많은 양의 세포 외 소포체를 단시간 내에 농축하고 세포 외 소포체의 손실을 줄일 수 있음을 발견하여 본 발명을 완성하게 되었다. The present inventors studied to develop a method for concentrating extracellular vesicles with high efficiency in a short time, and concentrated a large amount of extracellular vesicles in a short time by controlling the use of specific pore size filter and fluid sample composition and elution buffer. The present invention has been completed by discovering that the loss of extracellular vesicles can be reduced.
본 발명의 목적은 유체 샘플로부터 단시간 내에 높은 효율로 세포 외 소포체를 농축하는 방법으로서, 구체적으로 평균 기공 크기가 20 내지 100 nm인 필터를 사용하고 유체 샘플 조성 및 용리 완충액의 사용법을 조절하여 세포 외 소포체를 고효율로 농축하는 방법에 관한 것이다.An object of the present invention is a method of concentrating extracellular vesicles from a fluid sample with high efficiency within a short time, specifically using a filter having an average pore size of 20 to 100 nm and adjusting the use of the fluid sample composition and the elution buffer to extracellularly. A method for concentrating endoplasmic reticulum with high efficiency.
본 발명의 세포 외 소포체 농축법을 이용할 경우 농축 공정을 간편화하면서 농축 시간을 줄이고, 높은 효율로 세포 외 소포체를 농축할 수 있다. 이는 종래 농축 방법에 비하여 시간과 비용을 절감하고 농축 효율을 높인 것으로서 세포 외 소포체에 적합한 경제적인 농축 방법을 제시한 것이다. 특히, 본 발명에서 사용한 필터, 유체 샘플 및 용리 완충액의 특성은, 특정 장비에 국한되지 않고 공지된 여러 장비에 적용하여 사용 가능하다는 점에서 유용하다.When the extracellular vesicle enrichment method of the present invention is used, the extracellular vesicles can be concentrated with high efficiency while reducing the concentration time while simplifying the concentration process. This suggests an economical enrichment method suitable for extracellular vesicles by reducing time and cost and increasing enrichment efficiency compared to conventional enrichment methods. In particular, the properties of the filters, fluid samples and elution buffers used in the present invention are useful in that they can be applied to a variety of known equipment without being limited to specific equipment.
도 1는 본 발명에서 사용하는 농축 피펫 팁을 나타낸 것이다.1 shows a concentrated pipette tip for use in the present invention.
도 2는 50 nm 필터를 사용한 방식에서 세포 외 소포체와 분리되는 배양액의 총 부피를 나타낸 것이다.Figure 2 shows the total volume of culture separated from the extracellular vesicles in a manner using a 50 nm filter.
도 3은 울트라필터(ultrafilter, ≤ 20 nm)를 사용한 방식에서 세포 외 소포체와 분리되는 배양액의 총 부피를 나타낸 것이다. Figure 3 shows the total volume of the culture separated from the extracellular vesicles in a manner using an ultrafilter (≤ 20 nm).
도 4은 필터의 종류에 따른 단위 부피당 농축된 세포 외 소포체 입자수를 나타낸 것이다. 배수(Fold)는 배양액(culture medium)과 비교하여 농축된 비율을 확인한 값이다.Figure 4 shows the number of extracellular vesicle particles concentrated per unit volume according to the type of filter. Fold is a value confirming the concentration ratio compared to the culture medium (culture medium).
도 5는 필터의 종류에 따라 수득한 세포 외 소포체의 총 입자수를 나타낸 것이다.Figure 5 shows the total particle number of the extracellular vesicles obtained according to the type of filter.
도 6은 용리 완충액의 종류에 따른 불순물 함유량을 상대적으로 나타낸 것이다.Figure 6 shows the relative impurity content according to the type of elution buffer.
도 7은 용리 완충액의 종류에 따른 불순물 크기를 측정한 그래프이다.7 is a graph measuring the impurity size according to the type of elution buffer.
도 8는 용리 완충액의 사용 횟수(1회, 3회)에 따른 세포 외 소포체의 수득 효율 (recovery efficiency)을 나타낸 그래프이다. 8 is a graph showing the recovery efficiency of the extracellular vesicles according to the number of times of use of the elution buffer (once, three times).
도 9는 용리 완충액의 사용 횟수(3회)는 고정하고, 필터 크기(≤ 20 nm, 50nm, 100nm 및 200nm)에 따른, 농축된 세포 외 소포체의 수득 효율 (Concentration EVs) 및 농축되지 않고 필터를 통과하여 배출된 세포 외 소포체 (Filtrared EVs)의 제거 효율을 함께 나타낸 그래프이다.FIG. 9 shows the filter efficiency without concentration and the efficiency of obtaining extracellular vesicles (Concentration EVs) according to filter size (≦ 20 nm, 50 nm, 100 nm and 200 nm) with fixed use count of elution buffer (3 times). It is a graph showing the removal efficiency of extracellular vesicles (Filtrared EVs) discharged through.
도 10은 본 발명의 농축 방법과 ExoQuick을 이용한 농축 방법으로 농축한 세포 외 소포체의 수득 효율을 비교한 그림이다. 10 is a graph comparing the yield efficiency of the extracellular vesicles concentrated by the concentration method of the present invention and the concentration method using ExoQuick.
도 11은 본 발명의 농축 방법과 ExoQuick을 이용한 농축 방법으로 농축한 세포 외 소포체의 모습을 확인한 이미지이다.11 is an image confirming the appearance of the extracellular vesicles concentrated by the concentration method of the present invention and the concentration method using ExoQuick.
도 12은 본 발명의 농축 방법과 ExoQuick을 이용한 농축 방법으로 농축한 세포 외 소포체의 크기를 비교한 그림이다.12 is a diagram comparing the size of the extracellular vesicles concentrated by the concentration method of the present invention and the concentration method using ExoQuick.
도 13은 본 발명의 농축 방법과 ExoQuick을 이용한 농축 방법으로 세포 외 소포체를 농축할 시 요구되는 비용을 나타낸 그림이다.Figure 13 is a diagram showing the cost required to concentrate the extracellular vesicles by the concentration method and ExoQuick concentration method of the present invention.
도 14는 유체 샘플 내에 계면활성제의 포함 농도(0%, 0.05%, 1%, 5% 및 10%)에 따른 세포 외 소포체의 수득 효율을 나타낸 그래프이다.FIG. 14 is a graph showing the efficiency of obtaining extracellular vesicles according to the inclusion concentration (0%, 0.05%, 1%, 5% and 10%) of surfactant in the fluid sample.
본 발명의 제1양태는 유체 샘플로부터 세포 외 소포체(Extracellular vesicle)를 농축하는 방법으로서, A first aspect of the invention is a method of concentrating extracellular vesicles from a fluid sample,
(i) 유체 샘플을 담는 용기;(i) a container containing a fluid sample;
(ii) 평균 기공 크기가 20 내지 100 nm인 필터, 개구부, 및 배출구를 포함하는 농축 피펫 팁; 및(ii) a concentrated pipette tip comprising a filter, an opening, and an outlet having an average pore size of 20 to 100 nm; And
(iii) 상기 농축 피펫 팁을 통해 유체 샘플을 흡입하고 농축된 세포 외 소포체를 농축 피펫 팁으로부터 회수하도록 구성된 농축 유닛;(iii) a concentrating unit configured to aspirate a fluid sample through the condensation pipette tip and to recover the condensed extracellular vesicles from the condensation pipette tip;
을 포함하는 장치를 이용하여,Using a device that includes,
상기 농축 피펫 팁을 통하여 (i)의 용기에 담긴 유체 샘플을 흡입하고, 농축 피펫 팁의 필터에 포집된 세포 외 소포체를 용리시키는 단계를 포함하는, 방법을 제공한다.Inhaling the fluid sample contained in the container of (i) through the concentrated pipette tip and eluting the extracellular vesicles collected in the filter of the concentrated pipette tip.
이하, 본 발명을 자세히 설명한다.Hereinafter, the present invention will be described in detail.
세포 외 소포체를 분리하는 원심 분리 방법을 대체하는 방법 중 하나는 폴리에틸렌글리콜(Polyethyleneglycol, PEG)이라는 폴리머를 이용하여 세포 외 소포체를 농축하는 방법으로서, 이는 PEG가 세포 외 소포체의 용해도를 낮춰 용출시키고 가라앉히는 침전 현상을 이용한다. PEG와 세포 외 소포체의 반응 시간을 높일수록 많은 양의 세포 외 소포체를 침전시키며, 보통 4시간에서 24시간의 반응 시간을 필요로 한다. PEG에 의한 용출 방식을 사용하는 대표적 제품으로는 ExoQuick이 알려져 있다. 본 발명의 세포 외 소포체 분리 방식은 ExoQuick과 비교해 분리량이 2배 가까이 높고 비용이 20배 이상 저렴하여 생산성의 측면에서 효율적이다.One of the alternatives to centrifugal separation of extracellular vesicles is to concentrate the extracellular vesicles using a polymer called polyethyleneglycol (PEG). Uses sedimentation phenomenon to sit down. Increasing the reaction time of PEG and extracellular vesicles precipitates a large amount of extracellular vesicles and usually requires a reaction time of 4 to 24 hours. ExoQuick is known as a representative product using a dissolution method by PEG. The extracellular vesicles separation method of the present invention is more efficient than the ExoQuick in terms of productivity, since the separation amount is nearly two times higher and the cost is more than 20 times cheaper.
침전 방식 외에 자주 활용되는 세포 외 소포체 분리 방식은 필터를 이용하는 방식이다. 필터 방식은 세포 외 소포체보다 기공 크기가 작거나 유사한 필터를 사용하여 세포 외 소포체를 걸러내는 방법이다. 세포 외 소포체는 필터에 걸러지고 그 외 단백질들은 배출되기에 유체의 속도에 따라 분리 속도를 조절할 수 있으며, 반응 시간을 요구하지 않아 분리 시간은 빠르다. 하지만, 필터에 결합하거나 빠져나가는 세포 외 소포체의 비율이 높아 최종 회수율은 높지 않은 한계점이 있는 것으로 생각되어 왔다. 본 발명에서는 기존의 필터 방식을 사용하여 이의 장점을 가지면서도, 필터 크기와 용리 완충액의 구성 성분을 조절함으로써, 필터에 결합하거나 빠져나가는 세포 외 소포체의 비율을 낮추어 최종 분리 효율을 높인 것이 특징이다.In addition to the precipitation method, an extracellular vesicle separation method that is frequently used is a method using a filter. Filtering is a method of filtering extracellular vesicles using a filter having a pore size or similar to that of extracellular vesicles. Extracellular vesicles are filtered out and other proteins are discharged so that the rate of separation can be controlled by the speed of the fluid, and the separation time is fast because no reaction time is required. However, due to the high proportion of extracellular vesicles that bind to or exit the filter, it has been considered that the final recovery is not high. In the present invention, while using the existing filter method has its advantages, by controlling the filter size and the constituents of the elution buffer, it is characterized by lowering the ratio of extracellular vesicles bound to or out of the filter to increase the final separation efficiency.
종래 문제점을 해결하기 위해, 본 발명자들은 저비용으로 단시간 내에 세포 외 소포체를 농축하는 방법을 개발하고자 연구하였으며, (i) 평균 기공 크기가 20 내지 100 nm인 필터를 사용하고, (ii) 트리스 용리 완충액(Tris elution buffer) 또는 피비에스 용리 완충액(PBS　elution buffer), 거품 형성용 가스가 포함된 용리 완충액을 반복 사용하여 용리하고, (iii) 세포 외 소포체가 함유된 유체 샘플에 계면활성제를 추가로 포함할 경우, 종래 농축법에 비해 시간과 비용을 줄이면서도 고효율로 세포 외 소포체를 농축할 수 있음을 확인하였으며, 본 발명은 이에 기초한다.In order to solve the conventional problems, the present inventors studied to develop a method for concentrating extracellular vesicles in a short time at low cost, (i) using a filter having an average pore size of 20 to 100 nm, (ii) Tris elution buffer Repeatedly eluting with (Tris elution buffer) or PBS® elution buffer, an elution buffer containing a gas for foam formation, and (iii) adding an additional surfactant to the fluid sample containing extracellular vesicles. In this case, it was confirmed that the extracellular vesicles can be concentrated with high efficiency while reducing time and cost compared to the conventional concentration method, and the present invention is based on this.
구체적으로, 본 발명은 필터와 용리 완충액을 이용한 세포 외 소포체의 농축 방법이다. 본 발명에서 이용하는 농축 장치는 (i) 유체 샘플을 담는 용기; (ii) 평균 기공 크기가 20 내지 100 nm인 필터, 개구부, 및 배출구를 포함하는 농축 피펫 팁; 및 (iii) 상기 농축 피펫 팁을 통해 유체 샘플을 흡입하고 농축된 세포 외 소포체를 농축 피펫 팁으로부터 회수하도록 구성된 농축 유닛;을 포함하는 장치이다. Specifically, the present invention is a method for concentrating extracellular vesicles using a filter and an elution buffer. The concentrating device used in the present invention comprises (i) a container containing a fluid sample; (ii) a concentrated pipette tip comprising a filter, an opening, and an outlet having an average pore size of 20 to 100 nm; And (iii) a concentrating unit configured to aspirate a fluid sample through the condensation pipette tip and to recover the condensed extracellular vesicles from the condensation pipette tip.
본 발명의 장치에서, 농축 피펫 팁은 용출 포트를 별도로 포함하는 것일 수 있다. 즉, 농축 피펫 팁에 포함된 개구부가 샘플의 흡입 및 용리 작용을 동시에 하는 것일 수 있으나, 농축 피펫 팁에 용출 포트가 추가로 포함되어 있는 경우 유체 샘플 및 용리액이 서로 다른 부분을 통해 흡입될 수 있다.In the device of the present invention, the condensation pipette tip may be a separate elution port. That is, although the opening included in the concentrated pipette tip may simultaneously perform suction and elution of the sample, when the concentration pipette tip further includes an elution port, the fluid sample and the eluent may be sucked through different portions. .
본 발명의 농축방법에 사용된 장치를 구체적으로 설명하면 다음과 같다. Referring to the device used in the concentration method of the present invention in detail.
도 1(a)는 농축 피펫 팁(100)을 나타낸 것이다. 구체적으로, 개구부(105), 및 필터(101)를 포함하는 농축 피펫 팁(100)을 나타낸다. 또한, 농축 피펫 팁(100)은 필터(101), 투과물 퍼지(107), 투과물 드로(109)의 포팅(103)을 포함한다. 농축 피펫 팁(100)에 도시된 연결부(113)는 농축 피펫 팁(100)이 농축 피펫 팁(100)의 동작을 위한 농축 유닛에 연결되게 한다.Figure 1 (a) shows the concentrated pipette tip 100. Specifically, a thickening pipette tip 100 comprising an opening 105 and a filter 101 is shown. Concentrated pipette tip 100 also includes a filter 101, a permeate purge 107, and a potting 103 of permeate draw 109. The connection 113 shown in the enrichment pipette tip 100 allows the enrichment pipette tip 100 to be connected to a enrichment unit for operation of the enrichment pipette tip 100.
도 1(b)는 3개의 포트가 포함된 연결부(113)를 나타낸 것이다. 연결부(11)의 구성으로는 투과물 퍼지(107)에 연결된 제1 포트(115), 필터(101)에 연결된 제2 포트(117) 및 투과물 드로(109)에 연결된 제3 포트(119)가 있다. 상기 연결부(113)를 통해 제1 포트(115), 제2 포트(117) 및 제3 포트(119)는 농축 유닛과 연결되고, 농축 피펫 팁(100)은 농축 유닛에 연결된다. 유체 샘플은 하부 개구부(105) 내로 흡입되어 제3 포트(119)를 통해 투과물 드로(109)에 연결된 펌프를 사용하여 필터(101)의 다공성 표면을 통과한다. 필터(101) 또는 다른 박막 필터는 건조한 친수성 필터, 글리세린이 채워진 친수성 필터, 또는 처음에는 공기가 통과할 수 있게 해주는 또 다른 유형의 필터이며, 액체와 접촉할 경우 액체를 통과시킨다. 즉, 공기는 하부 개구부(105)로 흡입되고, 유체 샘플이 하부 개구부(105)로 흡입되어 필터(101)와 접촉하여 다공질 표면을 통과할 때까지 필터(101)의 다공질 표면을 통과한다.Figure 1 (b) shows a connection portion 113 including three ports. The connection part 11 includes a first port 115 connected to the permeate purge 107, a second port 117 connected to the filter 101, and a third port 119 connected to the permeate drawer 109. There is. The first port 115, the second port 117, and the third port 119 are connected to the concentration unit through the connection portion 113, and the concentration pipette tip 100 is connected to the concentration unit. The fluid sample is sucked into the lower opening 105 and passes through the porous surface of the filter 101 using a pump connected to the permeate draw 109 through the third port 119. The filter 101 or other thin film filter is a dry hydrophilic filter, a hydrophilic filter filled with glycerin, or another type of filter that allows air to pass through at first, and passes the liquid upon contact with the liquid. That is, air is sucked into the lower opening 105 and the fluid sample passes through the porous surface of the filter 101 until it is sucked into the lower opening 105 and contacts the filter 101 and passes through the porous surface.
본 발명에서 사용된 농축 시스템은 다음과 같다. 일회용 농축 피펫 팁(100)이 농축 유닛에 결합하는 튜브 모두가 농축 피펫 팁(100)의 상단에 위치한 단일 연결점에서 연결된다. 농축 유닛과 유체 샘플을 포함한 시스템과 함께 농축 피펫 팁(100)이 기능한다. 농축 피펫 팁(100)의 개구부(105)가 적절한 샘플 용기 내에 포함된 유체 샘플에 침지하여 농축 유닛이 작동한다. 이어서 유체 샘플이 농축 피펫 팁(100)에 흡입되고 필터(101)와 접촉하게 된다. 액체는 필터(101)를 통과하는 한편, 필터(101)의 기공 크기와 유사하거나 큰 입자는 필터(101)에 포집되어 유지된다. 모든 샘플이 필터(101)를 통과한 후 유체를 제거하여 포집된 샘플만 남게 되면, 농축 피펫 팁(100)의 하부 개구부(105)를 적절한 용기에 담그고 용리액을 이용하여 포집된 물질을 용리시킴으로써, 농축된 세포 외 소포체를 수득한다.The concentration system used in the present invention is as follows. All of the tubes to which the disposable thickening pipette tip 100 couples to the thickening unit are connected at a single connection point located at the top of the thickening pipette tip 100. The concentration pipette tip 100 functions with the system including the concentration unit and the fluid sample. The concentrating unit operates by immersing the opening 105 of the condensing pipette tip 100 in a fluid sample contained in a suitable sample container. The fluid sample is then sucked into the concentrated pipette tip 100 and brought into contact with the filter 101. The liquid passes through the filter 101, while particles similar to or larger than the pore size of the filter 101 are collected and retained in the filter 101. After all samples have passed through the filter 101, the fluid is removed to leave only the collected sample, by dipping the lower opening 105 of the concentrated pipette tip 100 into a suitable container and eluting the collected material using an eluent, Obtained concentrated extracellular vesicles.
본 발명의 세포 외 소포체를 농축하는 방법은 상기 농축 피펫 팁을 통하여 유체 샘플을 흡입하고, 농축 피펫 팁의 필터에 포집된 세포 외 소포체를 용리시키는 단계를 포함한다.The method for concentrating the extracellular vesicles of the present invention comprises aspirating a fluid sample through the concentrated pipette tip and eluting the extracellular vesicles collected in the filter of the concentrated pipette tip.
상기 유체 샘플은 0.05 내지 5 중량%의 계면활성제를 추가로 포함하는 것일 수 있다. 구체적으로, 계면활성제를 0.1 내지 4 중량%, 0.3 내지 3 중량%, 0.5 내지 2 중량%, 0.75 내지 1.5 중량%, 보다 구체적으로 1 중량% 포함하는 것일 수 있다. 이와 같이 유체 샘플에 계면활성제를 추가로 포함하는 경우, 필터에 결합된 세포 외 소포체가 쉽게 분리되어 농축되도록 유도하여, 세포 외 소포체 분리 효율을 향상 시킬 수 있다.The fluid sample may further comprise 0.05 to 5% by weight of surfactant. Specifically, the surfactant may include 0.1 to 4% by weight, 0.3 to 3% by weight, 0.5 to 2% by weight, 0.75 to 1.5% by weight, more specifically 1% by weight. As such, when the fluid sample further includes a surfactant, the extracellular vesicles bound to the filter can be easily separated and concentrated, thereby improving the efficiency of the extracellular vesicles separation.
본 발명의 계면활성제는 폴록사머, 폴리소르베이트, 또는 이들의 조합인 것일 수 있다. 그 예로, 계면활성제는 폴록사머 188, 폴리소르베이트 20 (Tween 20), 폴리소르베이트 40 (Tween 40), 폴리소르베이트 60 (Tween 60), 폴리소르베이트 80 (Tween 80) 및/또는 이들의 조합으로 이루어진 군에서 선택되는 것을 특징으로 한다. 본 발명의 실시예에서는 대표적인 계면활성제로 트윈 20을 사용하여 실시하였다.The surfactant of the present invention may be poloxamer, polysorbate, or a combination thereof. For example, the surfactant may be poloxamer 188, polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 80 (Tween 80) and / or their It is characterized in that it is selected from the group consisting of a combination. In the embodiment of the present invention it was carried out using Tween 20 as a representative surfactant.
상기 용리는 트리스 용리 완충액(Tris elution buffer) 또는 피비에스 용리 완충액(PBS　elution buffer)을 포함하는 용리 완충액을 사용하여 수행되는 것일 수 있다. 또한, 용리 횟수는 1회 내지 5회일 수 있다. The elution may be performed using an elution buffer comprising a Tris elution buffer or a PBS elution buffer. In addition, the number of elution may be 1 to 5 times.
또한, 상기 용리 완충액은 거품 형성용 가스를 추가로 포함할 수 있고, 보다 구체적으로 이산화탄소, 질소, 아르곤, 공기, 액화석유가스, 또는 이들의 조합을 포함할 수 있다. In addition, the elution buffer may further include a bubble forming gas, more specifically carbon dioxide, nitrogen, argon, air, liquefied petroleum gas, or a combination thereof.
계면활성제가 포함된 유체 샘플과 거품 형성용 가스가 포함된 용리 완충액이 혼합되는 경우, 상기 기체는 계면활성제와 반응하여 미세 방울을 만들어 마주치는 필터의 부피를 증가시킴으로써 빈 공간 없이 용리 완충액이 필터의 세포 외 소포체를 용리하도록 유도한다.When a fluid sample containing a surfactant is mixed with an elution buffer containing a gas for foaming, the gas reacts with the surfactant to form microdroplets, increasing the volume of the filter that is encountered so that the elution buffer is free from the filter. Induce extracellular vesicles to elute.
본 발명의 농축방법에서, 세포 외 소포체의 포집에 사용되는 필터(101)의 평균 기공 크기는 20 내지 100 nm, 보다 구체적으로 30 내지 80 nm, 40 내지 60 nm, 또는 50 nm일 수 있다. 이는 세포 외 소포체를 보다 효과적으로 포집하기 위한 적절한 크기이다.In the concentration method of the present invention, the average pore size of the filter 101 used for collecting extracellular vesicles may be 20 to 100 nm, more specifically 30 to 80 nm, 40 to 60 nm, or 50 nm. This is an appropriate size to more effectively capture extracellular vesicles.
세포 외 소포체의 크기는 20 nm 내지 1 um로 알려져 있다. 평균 20 nm 미만의 기공 크기를 가진 필터를 사용할 경우, 세포 외 소포체를 제외한 기타 액체나 단백질들이 필터를 빠져나가는 시간이 길어지거나 혹은 필터를 막게 되어 세포 외 소포체 농축이 진행되지 않는 어려움이 있다. 또한, 필터의 평균 기공 크기가 세포 외 소포체보다 큰 경우에 있어서는 세포 외 소포체가 손실되는 비율이 증가하게 되는 문제점이 있다. 특히, 필터의 평균 기공 크기가 100 nm 이상일 경우, 세포 외 소포체 중 20 내지 100 nm 정도의 크기를 가지는 작은 세포 외 소포체(엑소좀)의 비율이 줄어들어 문제가 된다.The size of the extracellular vesicles is known from 20 nm to 1 um. When a filter having an average pore size of less than 20 nm is used, other liquids or proteins except the extracellular vesicles may have a long time to pass through the filter or the filter may be blocked, thereby preventing the extracellular vesicles from being concentrated. In addition, when the average pore size of the filter is larger than the extracellular vesicles, there is a problem that the rate at which the extracellular vesicles are lost increases. In particular, when the average pore size of the filter is more than 100 nm, the proportion of small extracellular vesicles (exosomes) having a size of about 20 to 100 nm in the extracellular vesicles is a problem.
따라서, 액체 및 단백질이 제거되는 효율을 높이면서 세포 외 소포체의 손실율을 줄이는 효율적인 회수를 위해서는, 최적의 기공 크기를 가지는 필터를 사용해야 한다. 본 발명에서는 회수되는 세포 외 소포체의 총량을 가장 높일 수 있는 효율적인 필터로서 20 내지 100 nm의 평균 기공 크기를 가지는 필터를 선별한 것이 특징이다. Therefore, in order to increase the efficiency of removing liquids and proteins while reducing the loss rate of extracellular vesicles, a filter having an optimal pore size should be used. In the present invention, it is characterized by selecting a filter having an average pore size of 20 to 100 nm as an efficient filter that can increase the total amount of extracellular vesicles recovered.
즉, 본 발명은 세포 외 소포체를 농축시키기에 적합한 평균 기공 크기를 가지는 필터의 평균 기공 크기, 유체 샘플의 조성 변화 및 용리 완충액의 조성, 용리 횟수 등의 조건을 결정하여, 최적의 농축 방법을 위한 조합을 찾아낸 것이다. That is, the present invention determines the conditions of the average pore size of the filter having an average pore size suitable for concentrating the extracellular vesicles, the composition change of the fluid sample and the composition of the elution buffer, the number of elution, etc. I found a combination.
또한, 본 발명의 필터의 표면적은 5 cm2 내지 20 m2일 수 있으나, 이에 제한되지 않는다. 20 내지 100 nm의 평균 기공 크기를 가지는 필터라면 표면적에 구애되지 않고 본 발명의 농축 방법에 적용 가능하다. In addition, the surface area of the filter of the present invention may be 5 cm 2 to 20 m 2 , but is not limited thereto. Any filter having an average pore size of 20 to 100 nm can be applied to the concentration method of the present invention regardless of the surface area.
본 발명의 농축방법에서, 필터(101)에 포집된 세포 외 소포체를 용리시키기 위한 용액으로는, 트리스 용리 완충액(TRIS elution buffer) 또는 피비에스 용리 완충액 (PBS elution buffer)을 사용할 수 있다 용리액을 사용하기 위해서는 용리액 내부의 작은 불순물(nanoparticle)의 농도를 확인 후 선별하여 이후 농축되는 세포 외 소포체에 섞이는 양을 최소로 줄이는 것이 중요하다. In the concentration method of the present invention, as a solution for eluting the extracellular vesicles collected in the filter 101, a tris elution buffer or PBS elution buffer can be used. In order to determine the concentration of small impurities (nanoparticle) inside the eluate, it is important to reduce the amount of mixing in the extracellular vesicles to be concentrated afterwards to a minimum.
또한 용리액의 사용 횟수는 세포 외 소포체의 용리 효율을 높이는데 중요하다. 본 발명에서는 상기 설명한 평균 기공 크기의 필터를 사용하면서, 용리액의 사용 횟수, 즉 용리 횟수가 1회 ~ 5회일 경우 세포 외 소포체를 최적의 효율로 분리할 수 있음을 확인하였다. 용리 횟수가 5회를 초과할 경우, 분리되는 세포 외 소포체의 양은 증가할 수 있으나, 세포 외 소포체의 농도를 희석시키거나 형태를 망가뜨리는 문제점이 나타날 수 있다.In addition, the number of eluents used is important to increase the elution efficiency of the extracellular vesicles. In the present invention, using the filter of the average pore size described above, it was confirmed that the extracellular vesicles can be separated with optimum efficiency when the number of eluents used, that is, the number of elutions is 1 to 5 times. If the number of elution is more than five times, the amount of extracellular vesicles to be separated may increase, but there may be a problem of diluting the concentration of the extracellular vesicles or breaking the shape.
본 발명의 세포 외 소포체(Extracellular　vesicle)는 세포에서 생성되어 세포 밖으로 분비되는 소포체로서, 세포에서 세포 외 환경으로 방출(분비)되는 다양한 기능의 단백질, 예컨대, 각종 성장인자(Growth factors), 케모카인(Chemokines), 사이토카인(Cytokines), 전사인자(Transcription factors), RNAs(mRNA, miRNA 등), 지질 등의 다양한 생체 분자가 이것이 유래된 세포의 세포막과 동일한 지질 이중층의 세포막으로 봉입된 입자 형태의 구조체를 의미한다. 엑소좀(Exosome), 마이크로베시클(Microvesicle), 마이크로파티클(Microparticle) 등을 포함하나, 이에 제한되는 것은 아니다. Extracellular vesicles (Extracellular vesicles) of the present invention is a endoplasmic reticulum produced in the cells and secreted out of the cell, proteins of various functions released from the cell to the extracellular environment, such as various growth factors (Growth factors), chemokines ( Various biomolecules such as chemokines, cytokines, transcription factors, RNAs (mRNA, miRNA, etc.), lipids, etc. Means. Exosomes, microvesicles, microparticles, and the like, but are not limited thereto.
농축된 세포 외 소포체는 해당 기술분야에서 통상적으로 사용되는 분석 방법에 적용될 수 있다. 예컨대, 면역분석법(Immunoassay), 중합효소 연쇄 반응(Polymerase chain reaction: PCR), 전기화학(Electrochemical), 마이크로어레이(Microarray), 유세포 분석(Flow cytometry), 바이오센서, 랩온어칩(Lab-on-a-chip) 및 빠른 성장 검출(Rapid growth based detection) 등을 포함하지만, 이에 제한되지 않는다. Enriched extracellular vesicles can be applied to assay methods commonly used in the art. For example, immunoassay, polymerase chain reaction (PCR), electrochemical, microarray, flow cytometry, biosensor, lab-on-chip a-chip) and rapid growth based detection, but are not limited thereto.
특히, 본 발명의 농축방법에서 주요한 기술적 특징은 필터의 종류, 용리 완충액의 종류 및 사용 횟수에 있다. 당해 기술분야에서 입수 가능한 필터를 이용한 다양한 농축 방법의 실시양태가 모두 본 발명의 범위에 포함된다. In particular, the main technical features in the concentration method of the present invention is the type of filter, the type of elution buffer, and the number of times of use. Embodiments of various concentration methods using filters available in the art are all within the scope of the present invention.
본 발명의 상기 방법은 세포 외 소포체를 분리하는 단계를 추가로 포함하는 것일 수 있다. 본 발명의 방법에 의해 용리된 세포 외 소포체는 공지된 다양한 세포 외 소포체 분리 방법에 추가로 결합되어 사용될 수 있으나, 이에 제한되지 않는다. 그 예로, 상기 세포 외 소포체 분리 방법은 폴리머 기반 분리법, 원심분리 기반 분리법, 농축 기반 분리법 및 여과 기반 분리법일 수 있으나, 이에 제한되지 않는다.The method of the present invention may further comprise the step of separating the extracellular vesicles. The extracellular vesicles eluted by the method of the present invention may be used in addition to, but are not limited to, various known extracellular vesicle separation methods. For example, the extracellular vesicle separation method may be, but is not limited to, polymer-based separation, centrifugation-based separation, concentration-based separation, and filtration-based separation.
이하, 본 발명을 하기 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited only to these examples.
실시예 1. 세포 배양과 배양액 획득Example 1 Cell Culture and Culture Media Acquisition
본 연구에서는 지방 줄기세포를 배양한 배양액(Culture medium)을 활용하였다.In this study, culture medium in which adipose stem cells were cultured was used.
구체적으로, 지방 줄기세포를 약 80~90%로 배양 접시에 배양한 후 혈청 대체물(Serum replacement, Gibco) 10%가 함유된 MEM-alpha(Gibco) 배지로 교체해 주었다. 이틀 후, 지방 줄기세포의 배양액을 튜브에 모아서 원심분리기(3,000g, 20분)로 죽은 세포를 가라앉혀주고 상층액을 분리하였다. 수득한 상층액을 하기 실시예에 활용하였으며, 이하 '배양액(Culture medium)'으로 지칭한다.Specifically, the fat stem cells were cultured in a culture dish at about 80-90% and then replaced with MEM-alpha (Gibco) medium containing 10% of serum replacement (Serum replacement, Gibco). Two days later, the cultured cells of the adipose stem cells were collected in a tube, the dead cells were allowed to settle by centrifuge (3,000 g, 20 minutes), and the supernatant was separated. The obtained supernatant was used in the following examples, hereinafter referred to as 'culture medium'.
실시예 2. 시간 경과에 따른 농축 부피 분석Example 2. Concentration Volume Analysis Over Time
필터 크기를 다르게 하였을 때, 시간 경과에 따라 필터를 통과한 배양액의 총 부피를 측정하고 도 2 및 도 3에 나타내었다. When the filter size was changed, the total volume of the culture solution passed through the filter over time was measured and shown in FIGS. 2 and 3.
50 nm 기공 크기의 필터를 사용한 경우, 필터의 막힘이 적고 시간이 경과함에 따라 필터를 통과하는 배양액의 총 부피가 꾸준히 증가하여 55 ml/6.5분의 평균 속도로 농축이 가능하였다. 또한, 최종적으로 농축 가능한 배양액 총량은 약 60 ml임을 확인하였다(도 2). 반면, 울트라필터를 사용한 경우, 최대 농축 가능한 배양액의 양이 약 30 ml에 불과하여 시간이 경과하여도 농축 부피가 일정 수준 이상으로는 증가하지 않았고, 최종적으로 27 ml/6.5분의 평균 속도로 농축한 것으로 계산되었다(도 3).When the filter of 50 nm pore size was used, the total volume of the culture liquid passing through the filter was steadily increased as the filter was less clogged and the concentration was increased to 55 ml / 6.5 minutes. In addition, it was finally confirmed that the total amount of the culture medium is about 60 ml (Fig. 2). On the other hand, when the ultrafilter was used, the maximum concentration of the culture medium was only about 30 ml, and the concentration of the concentrate did not increase over a certain level even after time, and finally, the concentration was reached at an average rate of 27 ml / 6.5 minutes. Was calculated (FIG. 3).
즉, 전체적인 농축 속도와 농축양을 비교할 때, 50 nm 기공 크기의 필터를 사용할 경우 약 2배 가까이 높은 효율을 나타냄을 알 수 있었다. 또한, 울트라필터 (≤ 20 nm)의 경우, 필터가 쉽게 막히는 현상을 확인하였다. In other words, when comparing the overall concentration rate and the amount of concentration, it was found that the efficiency of about 2 times higher when using a filter having a pore size of 50 nm. In addition, in the case of the ultra filter (≤ 20 nm), it was confirmed that the filter clogging easily.
실시예 3. 단위 부피당 함유된 세포 외 소포체의 개수 확인 (농축 정도 확인)Example 3. Confirmation of the number of extracellular vesicles contained per unit volume (concentration check)
30 ml의 배양액을 50 nm 필터 또는 울트라필터를 통하여 흡입한 후, 트리스 용리 완충액으로 한번 용출하였다. 각각의 용출된 농축액 내의 세포 외 소포체의 농도를 Nanoparticle tracking analysis 장비(Malvern, LM10)로 측정하였다. 30 ml of culture was aspirated through a 50 nm filter or ultrafilter and then eluted once with Tris elution buffer. The concentration of extracellular vesicles in each eluted concentrate was measured with a Nanoparticle tracking analysis instrument (Malvern, LM10).
도 4에 나타난 바와 같이, 단일 부피당 존재하는 세포 외 소포체의 개수가 농축 전 배양액의 약 38배인 것을 확인하였다. 30 ml의 배양액을 울트라필터를 사용하여 농축한 경우, 단일 부피당 존재하는 세포 외 소포체의 개수가 배양액의 약 32배인것으로 확인되었다. 이를 통해, 50 nm 기공 크기의 필터를 사용할 경우, 울트라필터에 비하여 더 높은 비율로 농축할 수 있음을 확인하였다.As shown in Figure 4, it was confirmed that the number of extracellular vesicles present per single volume is about 38 times the culture before concentration. When 30 ml of the culture was concentrated using an ultrafilter, it was confirmed that the number of extracellular vesicles present per single volume was about 32 times that of the culture. Through this, it was confirmed that when using a filter of 50 nm pore size, it can be concentrated at a higher ratio than the ultra filter.
실시예 4. 회수된 세포 외 소포체의 총 개수 확인Example 4. Confirmation of the total number of extracellular vesicles recovered
10 ml의 배양액을 50 nm 필터 또는 울트라필터를 통하여 흡입한 후 트리스 용리 완충액을 사용하여 한번 용출하였다. 각각의 용출된 농축액 내의 세포 외 소포체의 농도를 Nanoparticle tracking analysis 장비(Malvern, LM10)로 측정하여 도 6에 나타내었다. 회수율(Recovery efficiency)은 배양액(culture medium)에 함유되어 있던 전체 세포 외 소포체 대비 분리한 세포 외 소포체의 비율을 나타낸 것이다.10 ml of culture was aspirated through a 50 nm filter or ultrafilter and eluted once with Tris elution buffer. The concentration of extracellular vesicles in each eluted concentrate was measured in a nanoparticle tracking analysis instrument (Malvern, LM10) and is shown in FIG. 6. Recovery efficiency represents the ratio of the extracellular vesicles separated from the total extracellular vesicles contained in the culture medium.
도 5에 나타난 바와 같이, 50 nm 필터를 사용한 경우 기존 배양액에 존재하는 세포 외 소포체의 70%를 회수하였지만, 울트라필터를 사용한 경우 55% 정도의 회수율이 나타남으로써, 50 nm 크기의 필터를 사용한 것과 비교하여 회수율이 현저히 낮아짐을 알 수 있었다.As shown in FIG. 5, when the 50 nm filter was used, 70% of the extracellular vesicles existing in the culture medium was recovered, but when the ultra filter was used, the recovery rate was about 55%. In comparison, the recovery was found to be significantly lower.
실시예 5. 용리 완충액에 따른 불순물 비교Example 5 Comparison of Impurities According to Elution Buffer
액상의 물질에는 세포 외 소포체와 유사한 크기의 불순물이 함유될 가능성이 높다. 따라서, 용리 완충액 내에 불순물의 존재를 확인 시험하였다. 각 용리 완충액 내의 불순물의 상대적 양과 크기를 Nanoparticle tracking analysis 장비(Malvern, LM10)로 측정하였다. Buffer 1과 3은 피비에스(PBS) 용리 완충액이고, Buffer 2와 4는 트리스(Tris) 용리 완충액이다.Liquid substances are likely to contain impurities of similar size to extracellular vesicles. Therefore, the presence of impurities in the elution buffer was tested for confirmation. Relative amounts and sizes of impurities in each elution buffer were measured by Nanoparticle tracking analysis equipment (Malvern, LM10). Buffers 1 and 3 are PBS elution buffers and Buffers 2 and 4 are Tris elution buffers.
도 6에 나타난 바와 같이, 동일한 용리 완충액일지라도 불순물이 함유된 정도가 다양하였다. 또한, 도 7에 나타낸 바와 같이 그 불순물들은 나노 크기인 20 nm부터 1 um까지 다양한 범위의 크기를 가지므로, 세포 외 소포체와 구분이 어려울 가능성이 높다. 따라서, 불순물이 적은 용리 완충액을 선별하여 시험을 수행하였다. As shown in FIG. 6, even the same elution buffer contained various impurities. In addition, as shown in FIG. 7, the impurities have a range of sizes ranging from 20 nm to 1 um, which is nanoscale, and thus, it is highly difficult to distinguish them from extracellular vesicles. Therefore, the test was performed by selecting an elution buffer containing few impurities.
실시예 6. 용리 완충액의 사용 횟수에 따른 총 수득률 증가 확인Example 6 Confirmation of Increase in Total Yield According to Number of Uses of Elution Buffer
10 ml의 배양액을 50 nm 필터에 흡입 후 트리스 용리 완충액을 한번 사용하여 필터에 묶여있는 세포 외 소포체를 용출한 제1그룹, 및 10 ml의 배양액을 50 nm 필터에 흡입 후 트리스 용리 완충액을 세번 사용하여 필터에 묶여있는 세포 외 소포체를 용출한 제2그룹을 대상으로, Nanoparticle tracking analysis 장비(Malvern, LM10)로 세포 외 소포체를 측정하여 회수율을 비교하였다. 회수율은 배양액(culture medium)에 함유되어 있던 전체 세포 외 소포체 대비 분리한 세포 외 소포체의 비율을 나타낸 것이다.Aspirate 10 ml of culture into a 50 nm filter and use the Tris elution buffer once to elute the extracellular vesicles bound to the filter, and inhale 10 ml of the culture into a 50 nm filter and use Tris elution buffer three times. In the second group eluting the extracellular vesicles bound to the filter by using the nanoparticle tracking analysis equipment (Malvern, LM10) by measuring the extracellular vesicles and compared the recovery rate. The recovery rate shows the ratio of the extracellular vesicles separated from the total extracellular vesicles contained in the culture medium.
도 8에 나타난 바와 같이, 용리 완충액을 한번 사용한 그룹에서는 회수율이 60%, 세번 사용한 그룹에서는 90%의 회수율이 나타났다. 즉, 용리 횟수를 3회로 증가시킴으로써 회수율이 높아진 것이다. As shown in Figure 8, the recovery was 60% in the group using the elution buffer once, 90% recovery in the group used three times. In other words, the recovery rate was increased by increasing the number of elutions to three times.
실시예 7. 다양한 필터의 크기에 따른 총 수득률 비교Example 7 Comparison of Total Yields with Various Filter Sizes
울트라필터(≤ 20 nm), 50 nm 필터, 100 nm 필터 및 200 nm 필터를 이용하여, 필터의 크기에 따른 총 수득률을 비교하였다.Ultrafilters (≦ 20 nm), 50 nm filters, 100 nm filters and 200 nm filters were used to compare the total yield according to the size of the filter.
10 ml의 배양액을 울트라필터, 50 nm 필터, 100 nm 필터 및 200 nm 필터에 흡입 후 트리스 용리 완충액을 세 번 사용하여 필터에 묶여있는 세포 외 소포체를 용출하였다. 농축된 세포 외 소포체를 측정할 뿐만 아니라 필터를 통과하여 농축되지 않고 배출된 세포 외 소포체도 함께 비교하였다. 각 세포 외 소포체를 Nanoparticle tracking analysis 장비(Malvern, LM10)로 측정하여 회수율을 비교하였다.10 ml of the culture was inhaled into the ultrafilter, 50 nm filter, 100 nm filter and 200 nm filter, and the extracellular vesicles bound to the filter were eluted using Tris elution buffer three times. Not only the concentrated extracellular vesicles were measured, but also the extracellular vesicles that had not been concentrated and passed through the filter were compared together. Each extracellular endoplasmic reticulum was measured by Nanoparticle tracking analysis equipment (Malvern, LM10) to compare the recovery.
도 9에 나타난 바와 같이, 50 nm 필터는 약 90%에 가까운 높은 회수율(검은색 그래프, Concentrated EVs)을 나타냈으며, 그 외 울트라 필터, 100 nm 필터 및 200 nm 필터는 약 50% 수준의 낮은 회수율을 나타냈다.As shown in FIG. 9, the 50 nm filter showed a high recovery (black graph, Concentrated EVs) close to about 90%, while the ultra filter, 100 nm filter and 200 nm filter had a low recovery of about 50%. Indicated.
이로부터, 100 nm 필터 및 200 nm 필터의 경우, 배출된 세포 외 소포체(흰색 그래프, Filtrated EVs)가 많음을 알 수 있었다. 또한, 울트라 필터의 경우 농축된 세포 외 소포체와 배출된 세포 외 소포체의 양을 합쳐도 기존 배양액에 존재하던 세포 외 소포체의 총 양에 한참 부족하였는데, 이는 울트라 필터 내부에 세포 외 소포체가 갇혀 용출되지도 필터를 통과하지도 못하는 것(점선 그래프, Trapped EVs)임을 알 수 있었다.From this, it was found that in the case of the 100 nm filter and the 200 nm filter, the extracellular vesicles discharged (white graph, Filtrated EVs) were large. In addition, the combined amount of extracellular vesicles and the extracellular vesicles concentrated in the ultra filter was far short of the total amount of the extracellular vesicles existing in the culture medium. It can be seen that it does not pass through the filter (dotted graph, Trapped EVs).
실시예 8. ExoQuick과의 총 수득률 비교Example 8 Comparison of Total Yield with ExoQuick
종래 알려진 PEG가 섞여있는 액체인 ExoQuick(제품명: ExoQuick-TCTM)을 이용한 농축방법과, 본 발명의 농축방법의 분리 효율을 비교하였다. The separation efficiency of the concentration method using ExoQuick (product name: ExoQuick-TC ™ ) which is a liquid mixed with known PEG, and the concentration method of the present invention were compared.
본 발명의 농축 방법의 경우, 10 ml의 배양액을 50 nm 필터에 흡입 후 트리스 용리 완충액을 세번 사용하여 용출하였다. 또한, 배양액 10 ml을 ExoQuick 2 ml과 섞어준 후 24시간 동안 배양액으로부터 세포 외 소포체가 용출되기를 기다렸다. 용출된 세포 외 소포체를 30분 동안 원심분리기(1500 g)에서 가라앉혀 분리하였다. 각각의 분리된 세포 외 소포체는 Nanoparticle tracking analysis 장비(Malvern, LM10)로 분석하였다.In the case of the concentration method of the present invention, 10 ml of the culture solution was aspirated into a 50 nm filter and eluted using Tris elution buffer three times. In addition, 10 ml of the culture was mixed with 2 ml of ExoQuick, and then waited for the extracellular vesicles to elute from the culture for 24 hours. Eluted extracellular vesicles were separated by sinking in a centrifuge (1500 g) for 30 minutes. Each isolated extracellular vesicles were analyzed by Nanoparticle tracking analysis equipment (Malvern, LM10).
도 10에 나타난 바와 같이, 50 nm 필터와 용리 완충액으로 3회의 용리를 거친 본 발명의 농축방법의 경우, 90.1% 효율로 세포 외 소포체를 분리하였다. 반면, ExoQuick을 사용한 경우 47.4%에 불과한 회수율을 확인하였다.As shown in FIG. 10, in the case of the concentration method of the present invention, which was eluted three times with a 50 nm filter and an elution buffer, extracellular vesicles were isolated at a 90.1% efficiency. On the other hand, when using ExoQuick, only 47.4% recovery was confirmed.
또한, Nanosight로 측정된 세포 외 소포체 이미지에서는 본 발명의 농축 방법에 따라 분리 된 세포 외 소포체가 배양액 및 Exoquick을 사용하여 분리 된 세포 외 소포체와 동일함을 알 수 있었으며(도 11), 세포 외 소포체의 크기 측정 결과에서도 본 발명의 농축 방법에 따라 분리된 세포 외 소포체의 크기와 배양액 및 Exoquick을 사용하여 분리 된 세포 외 소포체의 크기가 유사함을 알 수 있었다(도 12). 이러한 결과는 본 발명의 농축 피펫은 기존 방식에 비해 분리 수율은 높이고, 세포 외 소포체에 변형 또한 주지 않음을 의미한다.In addition, the extracellular vesicle images measured by Nanosight showed that the extracellular vesicles separated according to the enrichment method of the present invention are identical to the extracellular vesicles separated using the culture medium and Exoquick (Fig. 11). In the measurement results of the size of the extracellular vesicles separated according to the concentration method of the present invention and the size of the extracellular vesicles separated using the culture medium and Exoquick was found to be similar (Fig. 12). These results indicate that the enrichment pipette of the present invention has higher separation yield and no modification to extracellular vesicles as compared to the conventional method.
ExoQuick은 소포품으로 ExoQuick kit 와 원심분리 장비를 필요로 한다. 농축 피펫의 경우, 소모품으로 필터와 용리 완충액이 필요하고 장비로는 펌프를 필요로 한다. 도 13에 나타낸 바와 같이, 소모품의 비용으로 비교해보면 60 ml의 배양액에서 세포 외 소포체를 농축하는 비용이 Exoquick은 약 60만원이고 농축 피펫은 약 4만원 이내 수준으로 확인되었다. ExoQuick에 비해 본 발명의 농축 피펫을 사용할 경우 최소 15배 가량 비용 절감할 수 있음을 확인하였다.ExoQuick is a package that requires an ExoQuick kit and centrifuge equipment. Concentrated pipettes require filters and elution buffers as consumables and pumps as equipment. As shown in Figure 13, when compared to the cost of consumables, the cost of concentrating extracellular vesicles in a 60 ml culture solution was confirmed to be about 600,000 won for Exoquick and about 40,000 won for the concentrated pipette. When using the concentrated pipette of the present invention compared to ExoQuick was confirmed that the cost can be reduced by at least 15 times.
실시예 9. 계면활성제 농도에 따른 세포 외 소포체의 수득률 비교Example 9 Comparison of Yield of Extracellular Vesicles According to Surfactant Concentration
유체 샘플에 포함된 계면활성제의 농도(0%, 0.05%, 1%, 5% 및 10%)에 따른 총 수득률을 비교하였다.Total yields were compared according to the concentration (0%, 0.05%, 1%, 5% and 10%) of the surfactant included in the fluid sample.
10 ml의 배양액에 tween 20을 0%, 0.05%, 1%, 5%, 10%로 만들어 주었다. 각각의 배양액을 50 nm 필터에 흡입 후 트리스 용리 완충액을 세 번 사용하여 필터에 묶여있는 세포 외 소포체를 용출하였고 이를 Nanoparticle tracking analysis 장비(Malvern, LM10)로 세포 외 소포체를 측정하여 회수율을 비교하였다. Tween 20 was made into 0%, 0.05%, 1%, 5%, and 10% in 10 ml of culture. Each culture solution was inhaled into a 50 nm filter, and the extracellular vesicles bound to the filter were eluted using Tris elution buffer three times. The extracellular vesicles were measured using a nanoparticle tracking analysis device (Malvern, LM10) to compare the recovery rates.
도 14에 나타난 바와 같이, 유체 샘플에 1%의 계면활성제가 포함된 경우, 가장 높은 세포 외 소포체 수득률을 나타냄을 확인하였다. 5% 및 10%의 계면활성제가 포함된 경우, 세포 외 소포체를 녹여서 파괴하기 때문에 사용하기 적절하지 않은 농도임을 알 수 있었다. 즉, 유체 샘플에 계면활성제를 추가로 포함하여 분리하는 방법은 세포 외 소포체 분리 효율을 증가 시킬 수 있지만, 이에 적절한 농도는 1% 내외임을 알 수 있었다.As shown in FIG. 14, when the fluid sample contained 1% of the surfactant, the highest extracellular vesicle yield was confirmed. When 5% and 10% of the surfactant was included, it was found that the concentration is not suitable for use because the extracellular vesicles are dissolved and destroyed. In other words, the separation method by adding a surfactant to the fluid sample can increase the separation efficiency of the extracellular vesicles, but the appropriate concentration was found to be about 1%.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (7)

  1. 유체 샘플로부터 세포 외 소포체(extracellular vesicle)를 농축하는 방법으로서, A method of concentrating extracellular vesicles from a fluid sample,
    (i) 유체 샘플을 담는 용기;(i) a container containing a fluid sample;
    (ii) 평균 기공 크기가 20 내지 100 nm인 필터, 개구부, 및 배출구를 포함하는 농축 피펫 팁; 및(ii) a concentrated pipette tip comprising a filter, an opening, and an outlet having an average pore size of 20 to 100 nm; And
    (iii) 상기 농축 피펫 팁을 통해 유체 샘플을 흡입하고 농축된 세포 외 소포체를 농축 피펫 팁으로부터 회수하도록 구성된 농축 유닛;(iii) a concentrating unit configured to aspirate a fluid sample through the condensation pipette tip and to recover the condensed extracellular vesicles from the condensation pipette tip;
    을 포함하는 장치를 이용하여,Using a device that includes,
    상기 농축 피펫 팁을 통하여 (i)의 용기에 담긴 유체 샘플을 흡입하고, 농축 피펫 팁의 필터에 포집된 세포 외 소포체를 용리시키는 단계를 포함하는, 방법.Inhaling the fluid sample contained in the container of (i) through the concentrated pipette tip and eluting the extracellular vesicles collected in the filter of the concentrated pipette tip.
  2. 제1항에 있어서, 상기 농축 피펫 팁은 용출 포트를 포함하는 것인, 방법.The method of claim 1, wherein the thickening pipette tip comprises an elution port.
  3. 제1항에 있어서, The method of claim 1,
    상기 용리는 트리스 용리 완충액(Tris elution buffer) 또는 피비에스 용리 완충액(PBS　elution buffer)을 포함하는 용리 완충액을 사용하여 수행되며, 용리 횟수는 1회 내지 5회인, 방법.Wherein the elution is performed using an elution buffer comprising Tris elution buffer or PBS elution buffer, and the number of elutions is 1 to 5 times.
  4. 제3항에 있어서, The method of claim 3,
    상기 용리 완충액은 이산화탄소, 질소, 아르곤, 공기, 액화석유가스 또는 이들의 조합을 포함하는 것인, 방법.The elution buffer comprises carbon dioxide, nitrogen, argon, air, liquefied petroleum gas, or a combination thereof.
  5. 제1항에 있어서,The method of claim 1,
    상기 필터의 표면적은 5 cm2 내지 20 m2인, 방법.The surface area of said filter is between 5 cm 2 and 20 m 2 .
  6. 제1항에 있어서,The method of claim 1,
    상기 유체 샘플은 0.05 내지 5 중량%의 계면활성제를 추가로 포함하는 것인, 방법.Wherein the fluid sample further comprises 0.05 to 5% by weight of surfactant.
  7. 제1항에 있어서,The method of claim 1,
    상기 방법은 세포 외 소포체를 분리하는 단계를 추가로 포함하는 것인, 방법.The method further comprises the step of isolating extracellular vesicles.
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