Method for concentrating biological substances

Abstract

To make it possible to recover / concentrate an organism-derived substance from a liquid sample quickly, and simply and highly efficiently.SOLUTION: A concentration method of an organism-derived substance comprises: a step of adding polymer crowder and gel forming polymer having reversible sol / gel conversion characteristics to a liquid sample containing the organism-derived substance and stirring the sample; a step of separating the liquid sample into a multi-layer including a polymer crowder layer made of a polymer crowder phase and a gel forming polymer layer made of a gel forming polymer phase by centrifugal separation; and a step of inducing gelation of the gel forming polymer phase; and a step of dividing and separating the gelated gel forming polymer layer.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for concentrating a biological substance, such as extracellular vesicles (EVs, for example, exosomes), in a simple manner. [Background technology]

[0002] Previously, technologies have been proposed to recover and concentrate biological substances (e.g., proteins, viruses, EVs, etc.) that are easily affected by environmental factors such as temperature, pH, and the presence of organic solvents from liquid samples while maintaining their biological activity.

[0003] Well-known techniques for recovering and concentrating biological substances include ultracentrifugation, ultrafiltration, size exclusion chromatography, and affinity purification, and an appropriate method is used depending on the sample characteristics, post-treatment, operation time, cost, etc. In addition, there are also polymer precipitation methods that utilize the excluded volume effect and partition methods that utilize aqueous two-phase systems (ATPS), which are recovery / concentration methods that do not require special equipment and can be carried out at low cost.

[0004] Regarding EVs, recovery / concentration kits using polymer precipitation methods, such as Total Exosome Isolation Reagent (Invitrogen) and the ExoQuick series (System Biosciences), are commercially available. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6932529 [Patent Document 2] Korean Patent No. 10-1761680 [Patent Document 3] Korean Patent No. 10-1745455 [Patent Document 4] Special Publication No. 2022-508122

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Non-licensed literature

[0006] [Non-licensed document 1] H. Shin et al., “High-yield isolation of extracellular vesicles using aqueous two-phase system”, (2015) Scientific Reports, 5: 13103 [Non-licensed document 2] J. Kim et al., “Isolation of High-Purity Extracellular Vesicles by Extracting Proteins Using Aqueous Two-Phase System”, (2015) PLOS ONE, DOI:10.1371 / journal.pone.0129760. [Non-licensed document 3] A. Torres-Bautista et al., “Characterization and optimization of polymer-polymer aqueous two-phase systems for the isolation and purification of CaCo2 cell-derived exosomes”, (2022) PLoS ONE 17(9): e0273243.

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[0007] Conventional methods for recovering and concentrating biological substances, such as polymer precipitation using the excluded volume effect and partitioning using an aqueous two-phase system, have the advantages of being relatively easy to scale up and down without requiring special equipment and being low cost.

[0008] However, although the former method is very simple to operate, in order to improve the recovery efficiency of the target substance, it requires a long incubation step in which the target substance is aggregated to a size that can be sedimented by centrifugation by the excluded volume effect, and a long centrifugation step in which even small aggregates are precipitated.

[0009] The latter method utilizes the rapid formation of two phases (in about a few tens of seconds) and can distribute the target substance between two phases (layers) in a short time, but requires a certain degree of training. In other words, unlike a two-phase system of water and an organic solvent, both phases are aqueous, so the interfacial tension between the phases is small, and the operation of separating only the phase that preferentially contains the target substance without disturbing the interface is extremely difficult.

[0010] In the latter case, in order to improve the recovery efficiency of the target substance, it is important to collect all dispersed droplets (microdroplets) and separate each phase into two layers. Although increasing the polymer concentration is effective in increasing the interfacial tension between the phases and promoting two-layer separation, increasing the polymer concentration also increases the viscosity of the aqueous solution, making it difficult to improve both the recovery efficiency and operability.

[0011] The present invention has been made in consideration of the above, and an object of the present invention is to provide a method for concentrating biological substances, which enables rapid, simple and highly efficient recovery / concentration of biological substances from a liquid sample. [Means for solving the problem]

[0012] In order to achieve the above object, a first aspect of the present invention is a method for concentrating a biological substance, the method comprising the steps of: adding a polymer crowder and a gel-forming polymer having a reversible sol / gel conversion property to a liquid sample containing the biological substance, and stirring the liquid sample; separating the liquid sample into multiple layers including a polymer crowder layer consisting of a polymer crowder phase and a gel-forming polymer layer consisting of a gel-forming polymer phase by centrifugation; inducing gelation of the gel-forming polymer layer; and separating the gelled gel-forming polymer layer. The biological material is an extracellular vesicle, the polymer crowder is a polymer having a molecular crowding effect, and is PEG having an average molecular weight of 8,000 to 35,000 and a final concentration of 5 to 15%, and the gel-forming polymer phase is a polymer capable of repeated sol / gel conversion, and is gelatin having an average molecular weight of 10,000 to 100,000 and a final concentration of 0.1 to 0.5%. It is characterized by:

[0013] A second aspect of the present invention is a method for concentrating a biological substance, the method comprising the steps of adding a polymer crowder and a gel-forming polymer phase having a reversible sol / gel conversion property to a liquid sample containing the biological substance and stirring the mixture; inducing gelation of droplets of the gel-forming polymer phase; separating the liquid sample into multiple layers including a polymer crowder layer composed of the polymer crowder phase and a gelled gel-forming polymer layer by centrifugation; and separating the gelled gel-forming polymer layer. The biological material is an extracellular vesicle, the polymer crowder is a polymer having a molecular crowding effect, and is PEG having an average molecular weight of 8,000 to 35,000 and a final concentration of 5 to 15%, and the gel-forming polymer phase is a polymer capable of repeated sol / gel conversion, and is gelatin having an average molecular weight of 10,000 to 100,000 and a final concentration of 0.1 to 0.5%. It is characterized by: Effect of the Invention

[0014] According to the present invention, there is provided a method for concentrating a biological substance, which is capable of recovering / concentrating a biological substance from a liquid sample quickly, easily and efficiently. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a diagram (Example 1) showing an example of a method for concentrating a biological substance according to an embodiment of the present invention, in which droplets are separated. [Diagram 2] FIG. 10 is a diagram (Example 2) illustrating an example of a method for concentrating a biological substance according to an embodiment of the present invention, in which microgel separation is performed. [Diagram 3] Graphs showing the results of Verification 1, using the gelatin-polyethylene glycol (PEG) ATPS of Example 2 as an example, showing that the polymer concentration range in which two-layer separation is possible is expanded to a low polymer concentration range by microgelling the gelatin phase, where FIG. (a) is for an ATPS consisting of gelatin 100k and PEG 35k, FIG. (b) is for an ATPS consisting of gelatin 100k and PEG 8k, and FIG. (c) is for an ATPS consisting of gelatin 20k and PEG 8k. [Figure 4] Graphs showing the results of verification 2, using the gelatin-PEG ATPS of Example 2 as an example, showing that by centrifuging after microgelation, a higher proportion of gelatin is separated as the bottom layer (precipitate), where Fig. (a) is for an ATPS consisting of gelatin 100k and PEG 35k, Fig. (b) is for an ATPS consisting of gelatin 100k and PEG 8k, Fig. (c) is for an ATPS consisting of gelatin 60k and PEG 8k, Fig. (d) is for an ATPS consisting of gelatin 20k and PEG 8k, and Fig. (e) is for an ATPS consisting of gelatin 10k and PEG 8k. [Diagram 5] This shows the results of verification 3, and is a graph showing that the EV recovery rate increases by microgelling the gelatin phase in the gelatin-PEG ATPS of Examples 1 and 2, in comparison with the conventional method. [Figure 6]This graph shows the results of verification 4, and is a graph showing, in comparison with the conventional method, that microgelling the gelatin phase in the gelatin-PEG ATPS of Example 2 increases the EV recovery rate while reducing the contamination rate of bovine serum proteins. [Figure 7] 13 shows the results of verification 5, and is a graph showing the EV recovery rate by the concentration method using the gelatin-PEG ATPS of Example 2 in comparison with the EV recovery rate by other concentration methods. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Hereinafter, a method for concentrating a biological substance according to an embodiment of the present invention will be described with reference to the drawings. Note that in the embodiment (mode), the drawings are merely schematic illustrations of the invention, and may differ from the actual embodiment.

[0017] Explanation of terms Before describing the method for concentrating a biological substance according to the embodiment, the terms used in the description of this specification will be described. In the description of this specification, unless otherwise specified, the terms are interpreted based on the description in this section.

[0018] Biological substances are a general term for substances collected from living organisms, including cells that compose the organism, vesicles secreted from the organism, and (polymer) compounds synthesized or decomposed within the organism. Specifically, they include cells, blood cells, viruses, organelles, extracellular vesicles, extracellular membrane vesicles, and proteins.

[0019] A liquid sample is a sample of a liquid component collected from cultured cells or an organism (individual). Specifically, in a cell culture system, the liquid components are conditioned medium and cell lysate, while liquid components collected from an animal individual include serum, plasma, lymph, urine, ascites, pleural fluid, cerebrospinal fluid, and interstitial fluid, and liquid components collected from a plant individual include fruit juice, interstitial fluid, xylem sap, and phloem sap.

[0020] Polymer crowding is a biophysical phenomenon that has attracted attention in recent years in materials science and bio-related fields. It promotes molecular association and folding by restricting the volume that molecules can occupy due to the excluded volume effect.

[0021] Here, a polymer having a molecular crowding effect, i.e., a polymer crowder, usually has a molecular weight of about 5,000 or more and a concentration of about 5% or more, and creates a polymer crowding state. Examples of polymer crowders include polyethylene glycol, polyvinyl alcohol (PVA), polyvinylpyrrolidone, dextran (DEX), and ficoll.

[0022] The polymer crowder used in this embodiment is preferably PEG or PVA. Although details will be described later, in order to achieve the object of the present invention, the average molecular weight of PEG is preferably 3,000 to 50,000, and more preferably 8,000 to 35,000.

[0023] Reversible gel-forming polymers are polymers that can be repeatedly converted into sol / gel under certain conditions by forming partial cross-links between / within polymers to form a meshwork structure (gel) that holds many water molecules inside. Examples of polymers that can be repeatedly converted into sol / gel include gelatin, alginate, and agarose.

[0024] The gel-forming polymer used in this embodiment is preferably gelatin or alginate. Gelatin and alginate are biocompatible polymers that can be decomposed in vivo, are highly safe, and are widely used not only for food and industrial purposes but also in the medical field.

[0025] The sol / gel conversion characteristics of gelatin are affected by the species of organisms and molecular weight of the raw material, but the gelatin used in this embodiment may be of any raw material, molecular weight, or isoelectric point as long as it has a freezing point and melting point between 4° C. and 37° C. (The freezing point of gelatin derived from cows or pigs is 23-30° C., and the melting point is about 30-35° C.) The molecular weight of gelatin is preferably 10,000 to 100,000 to achieve the object of the present invention, as will be described in detail later.

[0026] An emulsion state is a state in which an aqueous two-phase system is suspended, causing the less liquid phase to be dispersed as droplets (or microgels) in the more liquid phase. The droplets (or microgels) may appear cloudy due to light scattering.

[0027] The process of separating droplets dispersed in a system into a single mass (usually a layer) due to the difference in specific gravity between the two phases is called two-phase separation. If the droplets remain dispersed, it is difficult to selectively collect only the droplets. Therefore, an efficient two-phase separation process (operation) is important.

[0028] The method for concentrating biological substances according to the present embodiment, which will be described later, is characterized by concentrating biological substances using an aqueous two-phase system that combines a polymer crowder with a high excluded volume effect and a reversible gel-forming polymer, and by separating and recovering biological substances by utilizing the sol / gel conversion properties.

[0029] Here, concentrating a biological substance means preparing a liquid containing a biological substance at a higher concentration than the liquid sample.

[0030] The method for concentrating a biological substance according to the present embodiment can be used as an initial purification step in a multi-step purification process. Specifically, the method includes a step of mixing and stirring a liquid sample containing a biological substance with a gel-forming polymer and a polymer crowder to prepare an emulsified aqueous two-phase system.

[0031] The stirring method is not particularly limited, and as long as an emulsion can be formed, stirring with a vortex mixer, stirring by pipetting, or mixing by inversion may be used.

[0032] The system includes at least a gel-forming polymer phase (gel-forming polymer layer) and a polymeric crowder phase (polymeric crowder layer). The polymeric crowder phase is a liquid phase mainly including a polymeric crowder, and may include a trace amount of a gel-forming polymer, but the concentration must be less than the gel-forming concentration to achieve the objectives of the present invention.

[0033] The gel-forming polymer phase is a liquid phase mainly containing a gel-forming polymer, and preferably does not actually contain a polymer crowder. Here, "does not actually contain a polymer crowder" means that it may contain a trace amount of polymer crowder to the extent that the action and effect of the present invention is not lost.

[0034] For the purposes of the present invention, it is preferred that droplets of the gel-forming polymer phase are dispersed in the polymer crowder phase.

[0035] In particular, until the two phases of the gel-forming polymer phase and the polymer crowder phase are formed, the gel-forming polymer must maintain a sol state without gelling as a single phase. That is, the final concentration of the gel-forming polymer must be set lower than the concentration at which a gel can be formed, and the concentration of the gel-forming polymer in the gel-forming polymer phase must be concentrated to a concentration at which a gel can be formed only after the formation of the two phases.

[0036] In addition, the final concentration of the system needs to be set so that the polymer crowder phase excludes biological substances and gel-forming polymers from the polymer crowder phase and concentrates the gel-forming polymer concentration in the gel-forming polymer phase to a concentration capable of gelling.

[0037] Therefore, it is desirable that the gel-forming polymer phase has a higher specific gravity than the polymer crowder phase, and the final concentration of the gel-forming polymer in the system is set so that the gel-forming polymer phase precipitates after the centrifugation step. For example, when the gel-forming polymer is gelatin, the final concentration of gelatin is preferably within the range of 0.05% to 5.0%, and more preferably 0.1 to 0.5%. On the other hand, when the polymer crowder is PEG, the final concentration of PEG is preferably within the range of 1 to 20%, and more preferably 5 to 15%.

[0038] The gel-forming polymer and the polymer crowder to be mixed with the liquid sample may be solid or liquid. The gel-forming polymer or the polymer crowder may be dissolved in a solvent and then mixed with the liquid sample.

[0039] The solvent is selected from water, saline, cell culture medium, buffer solution (eg, phosphate buffered saline (PBS)), etc., depending on the biological material to be concentrated.

[0040] In this embodiment, the step of preparing an emulsified aqueous two-phase system is followed by a step of gelling the gel-forming polymer phase, which is carried out according to the sol / gel conversion characteristics of the gel-forming polymer, for example, by cooling (below the gelling temperature, 0°C or higher) for gelatin, or by increasing the calcium ion concentration for alginate.

[0041] In order to achieve the object of the present invention, it is necessary to maintain the gelled state (gelling conditions) until the phase preferentially containing the biological substance is separated.

[0042] In this embodiment, the process after gelling the gel-forming polymer phase includes a process of recovering the gel-forming polymer phase. The method of recovering the gel-forming polymer phase is not limited as long as the gel-forming polymer phase can be obtained by separating all or a part of the gel-forming polymer phase from the polymer crowder phase. That is, the recovery method includes, for example, removing the polymer crowder phase by suction or decanting after separating the gel-forming polymer phase and the polymer crowder phase into two layers, and leaving only the gel-forming polymer phase.

[0043] In this embodiment, a step of centrifugal separation may be included to promote separation into two layers of the system. For example, when the gel-forming polymer phase is separated as droplets, a step of centrifugation may be inserted between the emulsification step and the gelation step of the gel-forming polymer phase, and when the gel-forming polymer phase is microgelled and collected, a step of centrifugation may be inserted between the microgelation step and the collection step.

[0044] The conditions for centrifugation depend on the size of the system, but the centrifugal acceleration may be in the range of 1,000 to 10,000xg and the time may be 1 to 10 minutes. In addition, the temperature is restricted to the gelling temperature when the gelling condition is temperature, as in the case of gelatin, but if there is no particular restriction, the temperature is preferably 4°C.

[0045] Furthermore, in this embodiment, after recovering the gel / microgel of the gel-forming polymer phase, a sol-inducing step of remelting the gel may be included. The sol-inducing step is performed according to the sol / gel conversion characteristics of the gel-forming polymer, for example, by heating (above the sol-inducing temperature, preferably below 37°C) in the case of gelatin, or by adding a calcium ion chelator (such as EDTA) to reduce the calcium ion concentration in the case of alginate. In order to prevent regelation of the gel-forming polymer phase, it may be appropriately diluted with a solvent.

[0046] Embodiment Below, we describe a simple and efficient method for recovering and concentrating biological materials by combining the exclusion of particles due to the excluded volume effect, which is one of the mechanisms of polymer precipitation, the rapid formation of two phases in an aqueous two-phase system, and the reversible sol / gel conversion property of a gel-forming polymer.

[0047] Example 1 FIG. 1 is a diagram for explaining a method for concentrating a biological substance according to this embodiment, taking droplet separation as an example.

[0048] First, as shown in Figure 1, a polymer crowder and a gel-forming polymer with reversible sol / gel conversion properties are added to a liquid sample containing biological substances and stirred (1). This creates an emulsion state (two-phase formation) in which droplets (sol) of the gel-forming polymer phase are dispersed in the polymer crowder phase. The polymer crowder phase always maintains a liquid state during the subsequent steps.

[0049] The polymer crowder phase and the gel-forming polymer phase are then separated into upper and lower bilayers by centrifugation (2-a).

[0050] Then, gelation of the gel-forming polymer is induced, and only the lower gel-forming polymer phase is gelled (3-a).

[0051] Thereafter, the lower gel-forming polymer phase (gel phase) and the upper polymer crowder phase (liquid phase) are separated and the gel-forming polymer phase is recovered (lower layer recovery) (4-a).

[0052] Finally, if necessary, the gel of the recovered gel-forming polymer phase is resolized / melted to obtain a liquid sample of concentrated biological material. (5) This sample may be diluted with a buffer solution (e.g., PBS) to reduce the concentration of the gel-forming polymer.

[0053] According to the present embodiment 1, biological substances can be concentrated from a liquid sample quickly and efficiently.

[0054] Example 2 FIG. 2 is a diagram for explaining the method for concentrating a biological substance according to this embodiment, taking microgel separation as an example.

[0055] First, as shown in Figure 2, a polymer crowder and a gel-forming polymer with reversible sol / gel conversion properties are added to a liquid sample containing biological substances and stirred (1). This results in an emulsion state (two-phase formation) in which droplets (sol) of the gel-forming polymer phase are dispersed in the polymer crowder phase. The polymer crowder phase always maintains a liquid state during the subsequent steps.

[0056] Next, gelation of the droplets is induced to prepare microgels of the gel-forming polymer phase (2-b).

[0057] Then, after the gel-forming polymer phase is microgelated, the microgel is precipitated from the polymer crowder phase (two-layer separation) by centrifugation (3-b).

[0058] Thereafter, the lower gel-forming polymer phase microgel (precipitate) and the upper polymer crowder phase (supernatant) are separated and the microgel is recovered (precipitate recovery) (4-b).

[0059] Finally, if necessary, the recovered gel-forming polymer phase microgels can be resolized / melted to obtain a liquid sample of concentrated biological material.(5) This sample can be diluted with a buffer solution (e.g., PBS) to reduce the concentration of the gel-forming polymer.

[0060] According to the second embodiment, biological substances can be concentrated from a liquid sample quickly and efficiently.

[0061] As described above, it takes time for biological substances rejected by a polymer crowder with a high excluded volume effect to aggregate to a size that can be precipitated by itself. However, by further adding a gel-forming polymer that can form a two-phase structure in combination with the polymer crowder, biological substances can be concentrated from a liquid sample quickly and efficiently.

[0062] That is, in Examples 1 and 2, when a biological substance having a high affinity for the gel-forming polymer is expelled (distributed) into the gel-forming polymer phase, droplets of the gel-forming polymer phase containing the biological substance are rapidly formed (rapidity of an aqueous two-phase system).

[0063] Moreover, even if the droplets are not incorporated into the gel-forming polymer phase, the formation of droplets in the gel-forming polymer phase restricts the space that can be occupied by the polymer crowder and the biological material (increasing the effective concentration), and furthermore, a large number of interfaces (droplet surfaces) that become aggregation sites for the biological material due to the excluded volume effect are formed, and the aggregation of the biological material on the interfaces is accelerated by repeated spontaneous fusion between the droplets, thereby quickly and efficiently promoting the aggregation of substances due to the excluded volume effect.

[0064] Furthermore, the weakness of aqueous two-phase systems, that is, the difficulty in separating the two phases due to the small interfacial tension between the phases, can be overcome by utilizing the properties of the gel-forming polymer (reversible sol / gel conversion property) to convert one phase into a gel phase (improved operability).

[0065] There are two possible times when the gelation induction step can be inserted: one is between the two-phase separation step and the phase separation step (Example 1), and the other is between the emulsification step and the two-phase separation step (Example 2). Either of these steps makes it easier to separate only the subsequent recovery phase.

[0066] In particular, in the latter case, the gel-forming polymer and droplets of the gel-forming polymer phase that remain dispersed in the polymer crowder phase and cannot be separated or recovered are easily separated or precipitated by gelation induction (improvement in the efficiency of two-phase formation and two-phase separation). This makes it possible to lower the optimal polymer concentration range that can be used in conventional aqueous two-phase systems, and the reduction in the viscosity of the system also leads to improved operability.

[0067] Examples A method for concentrating EVs as a biological substance using gelatin as a gel-forming polymer and PEG as a polymer crowder will be specifically described below. Note that the aspects of this embodiment are not limited to this specific example.

[0068] As mentioned above, gelatin can be combined with a polymer crowder such as PEG or PVA to create an aqueous two-phase system. By adjusting the type and molecular weight of both polymers and the final concentration of the entire system, a state in which the gelatin phase is dispersed as droplets in the system can be created.

[0069] For example, gelatin with average molecular weights of 10,000, 20,000, 60,000, and 100,000 (manufactured by Nitta Gelatin, all with an isoelectric point of 5) was swollen in PBS and stirred for 1 hour while heating at 37° C. Then, a 5% (w / w) gelatin 10k solution, a 5% (w / w) gelatin 20k solution, a 5% (w / w) gelatin 60k solution, and a 5% (w / w) gelatin 100k solution were prepared.

[0070] Similarly, for example, PEG (MP Biomedicals) with an average molecular weight of 8,000, PEG (Sigma-Aldrich) with an average molecular weight of 35,000, and DEX (MP Biomedicals) with an average molecular weight of 50,000 were swollen in PBS and thoroughly stirred at room temperature to prepare a 20% (w / w) PEG 8k solution, a 20% (w / w) PEG 35k solution, and a 10% (w / w) DEX 50k solution.

[0071] In response to this, human adipose-derived mesenchymal stem cell (hADSC) conditioned medium (MEM-α; GIBCO) / culture supernatant was subjected to two steps of low-speed centrifugation (4°C, 2,000xg, 20 min, and 4°C, 10,000xg, 30 min, respectively) to remove contaminating dead cells and cell fragments. After filtration through a 0.22-μm filter, EVs were precipitated by ultracentrifugation (4°C, 200,000xg, 1 h), resuspended in PBS to remove contaminating conditioned medium-derived proteins, and then subjected to further ultracentrifugation (4°C, 200,000xg, 1 h) to recover purified EVs.

[0072] EVs suspended in PBS were mixed with the labeling reagent ExoSparkler Exosome Protein Labeling Kit-Deep Red (Dojindo Laboratories; hereafter referred to as ExoSparkler; prepared according to the kit manual) and labeled by reacting at 37°C for 30 minutes.

[0073] After labeling, the unreacted label was removed using a size-exclusion chromatography column (PD-10; Cytiva, using PBS as buffer) to obtain the ExoSparkler-labeled EV sample (excitation / emission wavelengths used for fluorescence measurement were 640 / 680 nm).

[0074] Hereafter, the ExoSparkler-labeled EV sample was used within a concentration range in which the amount of ExoSparkler-labeled EV sample and the fluorescence intensity of the ExoSparkler label were in a linear relationship.

[0075] Test 1 Here, preliminary experiments were carried out to verify the improvement of two-phase separation ability and the expansion of the polymer concentration range in an aqueous two-phase system by the microgelation of droplets.

[0076] In this verification 1, first, a PEG solution, a gelatin solution, and PBS were mixed in a 1.5 mL microtube to prepare a system (1 mL) with an arbitrary polymer concentration, and the system was thoroughly stirred with a vortex mixer.

[0077] The mixture was then centrifuged at 10,000×g for 10 minutes using a tabletop centrifuge set at 37° C., and visual inspection was performed to see whether a gelatin phase layer (sol) had separated from the bottom of the tube.

[0078] If separation of the gelatin phase (layer) was not confirmed, the system was again thoroughly stirred with a vortex mixer, allowed to stand at 37°C for 10 minutes, and then cooled in a tube on ice for 10 minutes.

[0079] Thereafter, the mixture was centrifuged at 10,000×g for 10 minutes using a tabletop centrifuge set at 4° C., and visual inspection was performed to see whether gelatin phase microgels had precipitated at the bottom of the tube.

[0080] Figure 3 shows the state where the final PEG concentration and the final gelatin concentration of the system are set as two axes, and (a) shows the results when the aqueous two-phase system consisting of gelatin and PEG was gelatin 100k-PEG 35k, (b) shows the results when gelatin 100k-PEG 8k, and (c) shows the results when gelatin 20k-PEG 8k.

[0081] In Figures 3(a) to 3(c), in the high concentration range (upper right region), it was possible to confirm that the gelatin phase separated into two layers by centrifugation even though it remained in the form of droplets (circles in the figures).

[0082] However, when the polymer concentration was decreased (moving to the lower left region), a concentration range emerged in which no separation into two layers was observed, even if the system became cloudy (two phases were formed) due to emulsification.

[0083] However, even within that concentration range, the gelatin phase could be precipitated from the PEG phase by microgelling it by cooling (■ in the figure).

[0084] The cross marks in the figure indicate the concentration range in which the solution remained in one phase even after microgelation and did not separate.

[0085] The above results indicate that the effect of microgelation on bilayer separation is more pronounced as the polymer molecular weight decreases.

[0086] In other words, by introducing the microgelation process, it is now possible to concentrate substances using aqueous two-phase systems, even when combining low-molecular-weight polymers that have previously been difficult to form into aqueous two-phase systems, or when combining polymers at lower concentrations, and at the same time, the issue of system viscosity associated with the use of polymers is alleviated.

[0087] Test 2 Here, preliminary experiments were carried out to verify the improvement of the two-phase separation ability and the change in gelatin distribution in an aqueous two-phase system due to the microgelation of droplets.

[0088] In this verification 2, first, the PEG solution, gelatin solution, and PBS were combined in a 1.5 mL microtube to prepare a pair of systems (1 mL each) consisting of gelatin (4 mg) with a final concentration of 0.4% and PEG with an arbitrary final concentration, and the systems were thoroughly stirred using a vortex mixer.

[0089] One system was centrifuged at 10,000 x g for 10 minutes in a tabletop centrifuge set at 37°C, then cooled on ice to gel the lower gelatin phase, and the upper PEG phase was transferred to a separate tube to separate the two layers.

[0090] The other system was left to stand at 37° C. for 10 minutes, then cooled in a tube on ice for 10 minutes, and centrifuged at 10,000×g for 10 minutes in a tabletop centrifuge set at 4° C. The supernatant (PEG phase) was transferred to a separate tube, and the microgel precipitate of the gelatin phase that had settled at the bottom of the tube and the PEG phase were separated.

[0091] The gel / microgel precipitate of the recovered gelatin phase was then converted into a sol by incubation at 37°C.

[0092] Figure 4 shows the biaxial relationship between the final PEG concentration and the distribution ratio of gelatin in the gelatin phase (gelatin distribution in the gelatin phase), where (a) shows the results when the aqueous two-phase system consisting of gelatin and PEG was gelatin 100k-PEG 35k. Similarly, (b) shows the results when gelatin 100k-PEG 8k, (c) shows the results when gelatin 60k-PEG 8k, (d) shows the results when gelatin 20k-PEG 8k, and (e) shows the results when gelatin 10k-PEG 8k.

[0093] Here, the protein concentrations in the gelatin phase and the PEG phase were quantified using a BCA protein assay kit (Thermo Scientific), and the proportion of gelatin recovered as gelatin gel or microgel precipitate out of the gelatin (4 mg) added to the system was calculated as the gelatin distribution in the gelatin phase.

[0094] As is clear from Figures 4(a) to 4(e), the distribution of gelatin in the gelatin phase after two-layer separation is affected by the polymer molecular weight, and the separation of the gelatin phase is promoted more with increasing molecular weight of the gelatin and the PEG.

[0095] Furthermore, even with the same polymer concentration, it was found that the gelatin (phase) could be separated from the PEG phase more efficiently by forming a microgel and then allowing it to settle (■ in the figure) rather than by separating the droplets into two layers (circle in the figure).

[0096] The above results indicate that microgelation of the droplets efficiently separates the droplets dispersed throughout the system from the other phase, promoting two-phase separation. It also indicates that it is possible to construct an aqueous two-phase system by combining low-concentration, low-molecular-weight polymers, which was previously difficult to achieve, and that the reduced viscosity improves operability.

[0097] Test 3 Here, we examined the effect of droplet microgelation on improving EV recovery in the enrichment method using gelatin-PEG ATPS.

[0098] In this verification 3, first, ExoSparkler-labeled EV sample, gelatin solution, PEG solution, DEX solution, and PBS were appropriately combined in a 1.5 mL microtube to prepare systems (1 mL) 1 to 8 with final polymer concentrations as shown in Figure 5.

[0099] Systems 1 to 6 were gelatin-PEG ATPS with 0.4% gelatin (gelatin molecular weights 100k, 60k, and 20k)-7% PEG 8k, system 7 was a PEG precipitation system with 7% PEG 8k, and system 8 was a PEG-DEX ATPS with 7% PEG 8k-1% DEX 50k.

[0100] Systems 1, 3, and 5 are gelatin-PEG ATPS prepared by droplet separation, and are thoroughly stirred in a vortex mixer as shown in Table 1. The gelatin phase is centrifuged as droplets (10,000xg for 10 minutes in a tabletop centrifuge set at 37°C), and the lower layer gelled on ice is collected from the PEG phase to prepare a sample.

[0101] Systems 2, 4, and 6 are gelatin-PEG ATPS prepared by microgel separation, which are vigorously mixed with a vortex mixer, incubated at 37°C for 10 minutes, and then cooled on ice for 10 minutes (microgelation) as shown in Table 1 below. After separation into two layers by centrifugation (10,000xg for 10 minutes in a tabletop centrifuge set at 4°C), the precipitated microgels are collected from the PEG phase to prepare samples.

[0102] System 7 is a system in which gelatin addition is omitted in order to show the effect of two-phase formation in systems 1 to 6, that is, a system equivalent to the PEG precipitation method, and is sampled by centrifugation (10,000xg for 10 minutes in a tabletop centrifuge set at 37°C) after stirring with a vortex mixer, and collecting the precipitate, as shown in Table 1 below. It should be noted that by combining the operations and conditions of the present invention, the incubation step is omitted and the centrifugation time is also shortened compared to the general PEG precipitation method system.

[0103] System 8 is a PEG-DEX ATPS prepared using a conventional aqueous two-phase system, which is sampled by mixing with a vortex mixer, centrifuging at 10,000 x g for 10 minutes in a tabletop centrifuge set at 4°C, and collecting the bottom layer, as shown in Table 1 below.

[0104] In addition, in the cases of systems 1 to 6 in which the collected samples were gelled, the samples were melted by heating (37° C.) and then appropriately diluted with PBS to prevent re-gelation.

[0105] [Table 1]

[0106] As shown in Figure 5, the EV recovery rate (the ratio of concentrated EVs to the amount of EVs added to the system) was estimated from the amount of fluorescence of the collected sample (excitation wavelength 640 nm, fluorescence wavelength 680 nm). It was found that, even in systems with the same composition, the EV recovery rate was improved in systems 2, 4, and 6, in which the gelatin phase was microgelated and then centrifuged, compared to systems 1, 3, and 5, in which the gelatin phase was centrifuged in a droplet state. This effect of improving the recovery rate by microgelation was particularly noticeable in systems 5 and 6, which combined low-molecular-weight polymers that are less likely to form two phases / separate into two layers.

[0107] Even when the same concentration and molecular weight of the polymer crowder were used, only a very small amount of EVs were recovered in system 7, where two phases were not formed, compared to systems 1 to 6. This demonstrates the usefulness of droplet / microgel formation in the concentration technique of the present invention.

[0108] If system 7 is considered to be a PEG precipitation system processed using a time-saving process similar to the aqueous two-phase system, its low recovery rate suggests the high time performance of the concentration method using an aqueous two-phase system (note the difference in EV recovery rate in the PEG precipitation system that underwent a sufficient incubation time and centrifugation process in verification 5 (Figure 7) described below).

[0109] Whether droplet separation or microgel separation, the gelatin-PEG systems 1 to 6 involving gelation of the gelatin phase have significantly improved workability (operability) compared with the conventional general PEG-DEX system 8 in which both the upper and lower layers are liquid phases.

[0110] In addition, in particular, systems 2, 4, and 6, which perform microgel separation, showed increased EV recovery rates compared to system 8, demonstrating that the concentration method of the present invention is superior in terms of operability and recovery efficiency to the conventional concentration method using ATPS.

[0111] Test 4 Here, we compared a concentration method using gelatin-PEG ATPS, which includes a microgelation process of the gelatin phase, with a conventional concentration method using PEG-DEX ATPS.

[0112] In this verification 4, first, in a 1.5 mL microtube, an ExoSparkler-labeled EV sample or a calf serum (FCS) sample filtered through a 0.22 μm filter was added to an appropriate combination of gelatin 20k solution, gelatin 100k solution, PEG 8k solution, DEX 50k solution, and PBS to prepare ATPS (1 mL).

[0113] The final concentration of gelatin was 0.4%, the final concentration of DEX was 1%, and the final concentration of PEG was 7% or 15%. In addition, a background (BKG) system was prepared without adding any labeled EV or FCS samples for FCS protein quantification.

[0114] For gelatin-PEG ATPS, in the concentration method using the gelatin-PEG ATPS (0.4% gelatin 100k-PEG 8k) of Example 2, the system was stirred using a vortex mixer, incubated at 37°C and on ice for 10 minutes each, and then centrifuged (4°C, 10,000xg, 10 minutes).

[0115] The supernatant (PEG phase) was then separated, and the resulting gelatin phase microgel precipitate was melted at 37°C, and PBS was added to make the total volume 100 µL.

[0116] On the other hand, in the concentration method using PEG-DEX ATPS (PEG 8k-1% DEX 50k), the system stirred using a vortex mixer was centrifuged (4°C, 10,000xg, 10 minutes), the upper layer (PEG phase) was separated, and the lower layer (DEX phase) was obtained.

[0117] EV recovery was measured based on the amount of ExoSparkler fluorescence labeled to EVs. FCS protein contamination was measured by quantifying the protein concentrations in the upper and lower layers of the FCS sample-added system and the BKG system using a BCA protein assay kit (Thermo Scientific), and correcting the FCS protein concentration by subtracting the protein concentration in the BKG system from the protein concentration in the FCS sample system.

[0118] The FCS protein contamination rate was estimated as the proportion of FCS protein incorporated into the lower layer out of the total FCS protein added to the system.

[0119] Figure 6 is a plot with two axes representing EV recovery rate (vertical axis) and FCS protein contamination rate (horizontal axis) for the concentration method of the present invention (Example 2) and the conventional concentration method using PEG-DEX ATPS.

[0120] In the conventional PEG-DEX system, when the PEG concentration is increased to increase the interfacial tension between the PEG and DEX phases and promote phase separation, not only does the viscosity of the system increase, making it difficult to separate the phases (layers), but the lateral spread of the plot for the PEG-DEX system (△ in Figure 6) indicates that the rate of FCS protein contamination incorporated into the concentrated EV sample increases with increasing PEG concentration.

[0121] In contrast, in the present invention, regardless of the gelatin molecular weight, the plots (●■〇□ in Figure 6) are concentrated in the upper left region independently of the PEG-DEX system plot region, indicating that the FCS protein contamination rate in the EV sample was kept at a lower level than the PEG-DEX system and the EV recovery rate was improved to a high level.

[0122] From the above results, it can be seen that the concentration method of the present invention, particularly the concentration method of Example 2 involving droplet microgelation, has the advantage of not only a higher EV recovery rate but also a higher ability to remove FCS proteins contained in conditioned medium, etc., compared to the conventional concentration method using ATPS.

[0123] Alternatively, concentration can be achieved by directly adding a polymer crowder and a reversible gel-forming polymer to the conditioned medium (culture fluid) / culture supernatant after cell culture to form multiple phases.

[0124] Test 5 Here, we compared the EV recovery rate of the enrichment method using gelatin-PEG ATPS, which includes a microgelation step of the gelatin phase, with that of other enrichment methods.

[0125] Figure 7 shows the results of verification 5, comparing the EV recovery rate of EVs recovered / concentrated by the concentration method described in Example 2 with the EV recovery rate of EVs recovered / concentrated by conventional methods.

[0126] To calculate the EV recovery rate, ExoSparkler-labeled EV samples were used that were recovered / concentrated by the following methods (for ultracentrifugation, a 10-mL system using a 10-mL centrifuge tube, and for the aqueous two-phase system and polymer precipitation method, a 1-mL system using a 1.5-mL microtube).

[0127] That is, in the concentration method using gelatin-PEG ATPS (0.4% gelatin 100k-7% PEG 8k) according to Example 2, the system was stirred with a vortex mixer, incubated at 37°C and on ice for 10 minutes each, and then centrifuged (4°C, 10,000xg, 10 minutes).The supernatant (PEG phase) was then separated, and the resulting gelatin phase microgel precipitate was melted at 37°C and PBS was added to make the volume 100μL.

[0128] On the other hand, in the concentration method using PEG-DEX ATPS (7% PEG 8k-1% DEX 50k), the system stirred using a vortex mixer was centrifuged (4°C, 10,000 x g, 10 minutes), the upper layer (PEG phase) was separated, and the lower layer (DEX phase) was obtained.

[0129] In the concentration method using polymer precipitation-TEI (Total Exosome Isolation Reagent, Invitrogen), the system was prepared according to the manual, incubated overnight at 4°C, and then centrifuged (4°C, 10,000 x g, 60 minutes) to obtain a precipitate.

[0130] In the PEG precipitation method (7% PEG 8k), the prepared system was incubated at 4°C overnight and then centrifuged (4°C, 10,000 xg, 60 minutes) to obtain a precipitate.

[0131] In the ultracentrifugation method, the liquid volume was adjusted to 10 mL with PBS, and then ultracentrifugation (4°C, 200,000 xg, 60 minutes) was performed to obtain a precipitate.

[0132] In contrast, in the size exclusion chromatography method, the liquid volume was adjusted to 0.5 mL with PBS, and then loaded onto a qEV original GEN 2 (IZON) column according to the manual, eluted with PBS, and the 2.2-3.4 mL fraction was collected as the sample.

[0133] As is clear from Figure 7, when the EV recovery rate was calculated from the amount of fluorescence of the ExoSparkler label for each sample obtained, the gelatin-PEG ATPS of Example 2 not only improved operability and reduced operation time, but also showed a high EV recovery rate compared to concentration methods using general PEG-DEX ATPS and polymer precipitation methods.

[0134] As described above, according to the present embodiment, by combining the exclusion of particles due to the excluded volume effect, which is one of the mechanisms of polymer precipitation, the rapid formation of two phases in an aqueous two-phase system, and the reversible sol / gel conversion property of the gel-forming polymer, it is possible to simply and efficiently recover / concentrate biological substances.

[0135] In other words, by adding to a liquid sample a polymer crowder and a gel-forming polymer that can form two phases in combination with the polymer crowder, it becomes possible to rapidly, easily, and highly efficiently concentrate biological substances from the liquid sample.

[0136] In this specification, the description is limited to an aqueous two-phase system containing one type of polymer crowder and one type of reversible gel-forming polymer, but an aqueous multiphase system (multilayer) to which one or more types of polymer crowder or one or more types of gel-forming polymer are further added may be used as long as it does not impede the achievement of the object of the present invention.

[0137] The above describes the aspects of the present invention by illustrating the embodiment, but this is only an example, and the scope of the invention described in the claims can be modified in various ways without departing from the gist of the invention.

Claims

1. A method for concentrating a biological substance, comprising the steps of: adding a polymer crowder and a gel-forming polymer having reversible sol / gel conversion properties to the liquid sample containing the biological material, and stirring the liquid sample; separating the liquid sample into multiple layers by centrifugation, the multiple layers including a polymeric crowder layer comprising a polymeric crowder phase and a gel-forming polymer layer comprising a gel-forming polymer phase; inducing gelation of said gel-forming polymer layer; Separating the gelled gel-forming polymer layer; Equipped with The biological material is an extracellular vesicle, The polymer crowder is a polymer having a molecular crowding effect, and is PEG having an average molecular weight of 8,000 to 35,000 and a final concentration of 5 to 15%; The gel-forming polymer phase is a polymer capable of repeated sol / gel conversion, has an average molecular weight of 10,000 to 100,000, and is gelatin at a final concentration of 0.1 to 0.5%. A method for concentrating a biological substance, comprising:

2. A method for concentrating a biological substance, comprising the steps of: adding a polymer crowder and a gel-forming polymer phase having reversible sol / gel conversion properties to the liquid sample containing the biological material, and stirring the liquid sample; inducing gelation of droplets of said gel-forming polymer phase; separating the liquid sample into multiple layers, including a polymeric crowder layer consisting of a polymeric crowder phase and a gelled gel-forming polymer layer, by centrifugation; Separating the gelled gel-forming polymer layer; Equipped with The biological material is an extracellular vesicle, The polymer crowder is a polymer having a molecular crowding effect, and is PEG having an average molecular weight of 8,000 to 35,000 and a final concentration of 5 to 15%; The gel-forming polymer phase is a polymer capable of repeated sol / gel conversion, has an average molecular weight of 10,000 to 100,000, and is gelatin at a final concentration of 0.1 to 0.5%. A method for concentrating a biological substance, comprising:

3. A method for concentrating a biological substance as described in claim 1 or 2, characterized in that it further comprises a step of inducing solation of the separated gel-forming polymer layer and melting it.

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