JP7638481B2 - Method for concentrating biological substances - Google Patents

Description

The present invention relates to a method for concentrating biological substances, such as extracellular vesicles (EVs, e.g., exosomes), in a simple manner.

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.

Well-known techniques for recovering and concentrating biological substances include ultracentrifugation, ultrafiltration, size exclusion chromatography, and affinity purification, with the most appropriate method being used depending on the sample characteristics, post-treatment, operation time, and cost. Other recovery and concentration methods that do not require special equipment and can be carried out at low cost include polymer precipitation using the excluded volume effect and partitioning using an aqueous two-phase system (ATPS).

Regarding EV, recovery/concentration kits using polymer precipitation methods are commercially available, such as Total Exosome Isolation Reagent (Invitrogen) and the ExoQuick series (System Biosciences).

Patent No. 6932529 Korean Patent No. 10-1761680 Korean Patent No. 10-1745455 Special Publication No. 2022-508122 International Publication No. 2019/160519 Patent No. 5271354

H. Shin et al., “High-yield isolation of extracellular vesicles using aqueous two-phase system”, (2015) Scientific Reports, 5: 13103 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. 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. Y. Weng et al., “Effective isolation of exosomes with polyethylene glycol from cell culture supernatant for in-depth proteome profiling”, (2016) Analyst, 141: 4640-4646 T. Nezu & H. Maeda., “Phase Separation Coupled with Gelation in Polyethylene Glycol-Gelatin-Aqueous Buffer System”, (1991) Bulll Chem. Soc. Jpn, 64: 1618-1622 M. Yanagisawa, et al., “Phase separation in binary polymer solution: Gelatin/Poly(ethylene glycol) system”, (2014) Journal of Molecular Liquids 200: 2-6

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 advantage that they do not require special equipment, are relatively easy to scale up and down, and are low cost.

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

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. That is, unlike a two-phase system of water and an organic solvent, both phases are aqueous, so the interfacial tension between the phases is low, and it is extremely difficult to separate only the phase that preferentially contains the target substance without disturbing the interface.

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

The present invention has been made in view of the above, and its object is to provide a method for concentrating biological substances that can quickly, easily, and efficiently recover and concentrate biological substances from liquid samples.

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 reversible sol/gel conversion properties to a liquid sample containing the biological substance, and stirring the mixture; separating the liquid sample by centrifugation 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; inducing gelation of the gel-forming polymer layer; and separating the gelled gel-forming polymer layer , wherein the biological substance 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% .

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 reversible sol/gel conversion properties 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 , wherein the biological substance 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% .

The present invention provides a method for concentrating biological substances that can quickly, easily, and efficiently recover and concentrate biological substances from liquid samples.

FIG. 1 is a diagram (Example 1) illustrating an example of a method for concentrating a biological substance according to an embodiment of the present invention, in which droplets are separated. FIG. 11 is a diagram (Example 2) showing an example of a method for concentrating a biological substance according to an embodiment of the present invention, in which microgel separation is performed. 1 is a graph showing the results of verification 1, and shows 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, using the gelatin-polyethylene glycol (PEG) ATPS of Example 2 as an example. FIG. 1(a) is for the ATPS consisting of gelatin 100k and PEG 35k, FIG. 1(b) is for the ATPS consisting of gelatin 100k and PEG 8k, and FIG. 1(c) is for the ATPS consisting of gelatin 20k and PEG 8k. 11 is a graph showing the results of verification 2, taking the gelatin-PEG ATPS of Example 2 as an example, showing that a higher ratio of gelatin is separated as a lower layer (precipitate) by centrifuging after microgelation, where FIG. 11(a) is for an ATPS consisting of gelatin 100k and PEG 35k, FIG. 11(b) is for an ATPS consisting of gelatin 100k and PEG 8k, FIG. 11(c) is for an ATPS consisting of gelatin 60k and PEG 8k, FIG. 11(d) is for an ATPS consisting of gelatin 20k and PEG 8k, and FIG. 11(e) is for an ATPS consisting of gelatin 10k and PEG 8k. 1 shows the results of verification 3, and is a graph showing that the EV recovery rate is increased by microgelling the gelatin phase in the gelatin-PEG ATPS of Examples 1 and 2, in comparison with the conventional method. This is a graph showing the results of verification 4, which is a comparison with the conventional method, showing that microgelling the gelatin phase in the gelatin-PEG ATPS of Example 2 increases the EV recovery rate and reduces the bovine serum protein contamination rate. 13 shows the results of Verification 5, and is a graph showing the EV recovery rate in the concentration method using the gelatin-PEG ATPS of Example 2 in comparison with the EV recovery rates in other concentration methods.

The method for concentrating biological substances according to the embodiment of the present invention will be described below with reference to the drawings. Note that in the embodiment (mode), the drawings are schematic diagrams showing the outline of the invention and may differ from the actual one.

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 explained. In the description of this specification, unless otherwise specified, the terms are interpreted based on the description in this section.

Biological substances are a general term for substances recovered from living organisms, including cells that make up living organisms, vesicles secreted from living organisms, and (polymeric) compounds synthesized or decomposed within living organisms. Specifically, they include cells, blood cells, viruses, organelles, extracellular vesicles, extracellular membrane vesicles, and proteins.

A liquid sample is a sample of liquid components collected from cultured cells or organisms (individuals). Specifically, in cell culture systems, liquid components include conditioned medium and cell lysates. Liquid components collected from individual animals include serum, plasma, lymph, urine, ascites, pleural fluid, cerebrospinal fluid, and interstitial fluid, while liquid components collected from individual plants include fruit juice, interstitial fluid, xylem sap, and phloem sap.

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 limiting the volume that molecules can occupy due to the excluded volume effect.

Here, a polymer with 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 crowded state of polymers. Examples of polymer crowders include polyethylene glycol, polyvinyl alcohol (PVA), polyvinylpyrrolidone, dextran (DEX), and ficoll.

The polymer crowder used in this embodiment is preferably PEG or PVA. Details will be described later, but 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.

A reversible gel-forming polymer is a polymer that can be repeatedly converted into a sol/gel state under certain conditions, forming partial cross-links between/within the polymer and constructing a meshwork structure (gel) that holds many water molecules inside. Examples of polymers that can be repeatedly converted into a sol/gel state include gelatin, alginate, and agarose.

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

The sol/gel conversion characteristics of gelatin are affected by the species of organism 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). Details will be described later, but in order to achieve the objectives of the present invention, the preferred molecular weight of gelatin is 10,000 to 100,000.

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, and the droplets (or microgels) may appear cloudy due to light scattering.

When droplets dispersed in a system are separated into a single mass (usually a layer) due to the difference in specific gravity between the two phases, this is called two-phase separation. If the droplets remain dispersed, it is difficult to selectively collect only the droplets. For this reason, an efficient two-phase separation process (operation) is important.

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 using the sol/gel conversion properties.

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

The method for concentrating a biological substance according to this embodiment can be used as the initial stage of purification in a multi-stage purification. Specifically, it includes a step of preparing an emulsified aqueous two-phase system by mixing and stirring a liquid sample containing a biological substance with a gel-forming polymer and a polymer crowder.

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

The system includes at least a gel-forming polymer phase (gel-forming polymer layer) and a polymer crowder phase (polymer crowder layer). The polymer crowder phase is a liquid phase that mainly contains a polymer crowder and may contain 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.

The gel-forming polymer phase is a liquid phase that mainly contains 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 small amount of polymer crowder to the extent that the action and effect of the present invention is not lost.

To achieve the objectives of the present invention, it is preferable that droplets of the gel-forming polymer phase are dispersed in the polymer crowder phase.

In particular, until the two phases of the gel-forming polymer phase and the polymer crowder phase are formed, the gel-forming polymer must remain in a sol state without gelling as a single phase. In other words, the final concentration of the gel-forming polymer must be set so that it is 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 is concentrated to a concentration at which a gel can be formed only after the formation of the two phases.

In addition, the final concentration of the system must be set so that the polymer crowder phase excludes the biological material and the gel-forming polymer from the polymer crowder phase and concentrates the gel-forming polymer concentration in the gel-forming polymer phase to a concentration that allows gelation.

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 settles 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%.

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

The solvent to be used is selected from water, saline, cell culture medium, and buffer solutions (e.g., phosphate-buffered saline (PBS)) that are suitable for the biological material to be concentrated.

In this embodiment, the process includes a step of gelling the gel-forming polymer phase after the step of preparing an emulsified aqueous two-phase system. The gelling step 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) in the case of gelatin, or by increasing the calcium ion concentration in the case of alginate.

To achieve the objectives of the present invention, it is necessary to maintain the gelled state (maintain the gelling conditions) until the phase that preferentially contains the biological substance is separated.

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

In this embodiment, a centrifugation step may be included to promote separation into two layers of the system. For example, if the gel-forming polymer phase is separated as droplets, a centrifugation step may be inserted between the emulsification step and the gelling step of the gel-forming polymer phase, and if the gel-forming polymer phase is microgelled and recovered, a centrifugation step may be inserted between the microgelling step and the recovery step.

The conditions for centrifugation depend on the size of the system, but the centrifugal acceleration should be in the range of 1,000 to 10,000 x g, and the time should 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 are no particular restrictions, it is preferably 4°C.

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.

Hereinafter, a simple and efficient method for recovering/concentrating biological substances will be described, which combines 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.

Example 1
FIG. 1 is a diagram for explaining a method for concentrating a biological substance according to the present embodiment, taking droplet separation as an example.

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 a biological substance 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 remains in a liquid state during the subsequent steps.

Then, the polymer crowder phase and the gel-forming polymer phase are separated into two layers, an upper layer and a lower layer, by centrifugation (2-a).

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

Then, 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).

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

According to this embodiment 1, biological substances can be concentrated from liquid samples quickly and efficiently.

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

First, as shown in Figure 2, for example, a polymer crowder and a gel-forming polymer with reversible sol/gel conversion properties are added to a liquid sample containing a biological substance 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.

The droplets are then induced to gel, preparing microgels of the gel-forming polymer phase (2-b).

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).

Then, the microgel (precipitate) of the gel-forming polymer phase in the lower layer and the polymer crowder phase (supernatant) in the upper layer are separated, and the microgel is recovered (precipitate recovery) (4-b).

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

According to this embodiment 2, biological substances can be concentrated from liquid samples quickly and efficiently.

As described above, it takes time for the biological material rejected by the polymer crowder, which has 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, the biological material can be concentrated from the liquid sample quickly and efficiently.

That is, in Examples 1 and 2, when a biomaterial that has 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 biomaterial are rapidly formed (rapidity of an aqueous two-phase system).

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 a large number of interfaces (surfaces of the droplets) that become aggregation sites for the biological material due to the excluded volume effect are formed. Furthermore, repeated spontaneous fusion between the droplets accelerates the aggregation of the biological material on the interfaces, thereby swiftly and efficiently promoting the aggregation of substances due to the excluded volume effect.

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).

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 way, it makes it easier to separate only the subsequent recovery phase.

In particular, in the latter case, gelation induction makes it easier for 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 to separate and precipitate (improving the efficiency of two-phase formation and two-layer separation). This makes it possible to lower the optimal polymer concentration range that can be used in conventional aqueous two-phase systems, and the reduced viscosity of the system also leads to improved operability.

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 aspect of the present embodiment is not limited to only this specific example.

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.

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 while heating at 37°C for 1 hour. 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.

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 each swollen in PBS and thoroughly stirred at room temperature. Then, a 20% (w/w) PEG 8k solution, a 20% (w/w) PEG 35k solution, and a 10% (w/w) DEX 50k solution were prepared.

In response to this, human adipose-derived mesenchymal stem cell (hADSC) conditioned medium (MEM-α; GIBCO)/culture supernatant was subjected to two stages of low-speed centrifugation (4°C, 2,000 x g, 20 min, and 4°C, 10,000 x g, 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, 200,000 x g, 1 h), resuspended in PBS to remove contaminating conditioned medium-derived proteins, and then subjected to further ultracentrifugation (4°C, 200,000 x g, 1 h) to recover purified EVs.

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 allowed to react at 37°C for 30 minutes to label them.

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

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.

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.

In this experiment 1, first, a system of any polymer concentration (1 mL) was prepared by combining the PEG solution, gelatin solution, and PBS in a 1.5 mL microtube, and then thoroughly mixing the solution with a vortex mixer.

The tube was then centrifuged at 10,000 x g for 10 minutes in 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.

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

Then, the mixture was centrifuged at 10,000 x g for 10 minutes in a tabletop centrifuge set at 4°C, and visually inspected to see whether gelatin phase microgels had precipitated at the bottom of the tube.

Figure 3 shows the state where the final PEG concentration and final gelatin concentration of the system are on two axes, with figure (a) showing the results when the aqueous two-phase system consisting of gelatin and PEG was gelatin 100k-PEG 35k. Similarly, figure (b) shows the results when the combination was gelatin 100k-PEG 8k, and figure (c) shows the results when the combination was gelatin 20k-PEG 8k.

In Figures 3(a) to 3(c), in the high concentration range (upper right region), the gelatin phase was separated into two layers by centrifugation even though it remained in droplet form (circles in the figures).

However, as the polymer concentration is decreased (moving to the lower left region), a concentration range appears where no separation into two layers is observed, even if the system becomes cloudy due to emulsification (two phases are formed).

However, even in this concentration range, the gelatin phase could be precipitated from the PEG phase by microgelling it through cooling (■ in the figure).

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

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

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 viscosity issues associated with the use of polymers is alleviated.

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.

In this experiment 2, first, the PEG solution, gelatin solution, and PBS were mixed 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 then thoroughly mixed using a vortex mixer.

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.

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

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

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), with (a) showing 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.

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.

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 the greater the molecular weight of the gelatin combined and the greater the molecular weight of the PEG.

In addition, 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 separating the droplets into two layers (circle in the figure).

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

Test 3
Here, we examined the effect of droplet microgelation on improving EV recovery in a concentration method using gelatin-PEG ATPS.

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 the final polymer concentrations shown in Figure 5.

Systems 1-6 were gelatin-PEG ATPS with 0.4% gelatin (gelatin molecular weights 100k, 60k, 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.

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 below. The gelatin phase is then centrifuged as droplets (10,000 x g 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.

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

System 7 is a system in which the addition of gelatin is omitted in order to demonstrate the effect of two-phase formation in systems 1 to 6, i.e., a system equivalent to the PEG precipitation method. As shown in Table 1 below, after stirring with a vortex mixer, centrifugation (10,000 xg for 10 minutes in a tabletop centrifuge set at 37°C) is performed, and the precipitate is collected to prepare a sample. 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.

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.

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

As shown in Figure 5, the EV recovery rate (proportion of concentrated EV recovered from the amount of EV added to the system) was estimated from the amount of fluorescence of the recovered sample (excitation wavelength 640 nm, fluorescence wavelength 680 nm). It was found that, even in systems with the same composition, systems 2, 4, and 6, in which the gelatin phase was microgelled and then centrifuged, had a higher EV recovery rate than systems 1, 3, and 5, in which the gelatin phase was centrifuged while still in droplet form. This effect of improving the recovery rate by microgelling 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.

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

If system 7 is considered to be a PEG precipitation system that has been processed using the same time-saving process as 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 sufficient incubation and centrifugation processes in verification 5 (Figure 7) described below).

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

In addition, in particular, systems 2, 4, and 6, which perform microgel separation, showed an increase in EV recovery rate compared to system 8, and the concentration method of the present invention was found to be superior in terms of operability and recovery efficiency to the concentration method using conventional ATPS.

Test 4
Here, a concentration method using gelatin-PEG ATPS, which includes a microgelation step of the gelatin phase, was compared with a conventional concentration method using PEG-DEX ATPS.

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).

The final gelatin concentration was 0.4%, the final DEX concentration was 1%, and the final PEG concentration was 7% or 15%. In addition, for FCS protein quantification, a background (BKG) system was also prepared in which neither the labeled EV sample nor the FCS sample was added.

In the case of 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).

Then, the supernatant (PEG phase) was separated, and the resulting gelatin phase microgel precipitate was melted at 37°C and PBS was added to make a volume of 100 μL.

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, 10,000 xg, 10 minutes), the upper layer (PEG phase) was separated, and the lower layer (DEX phase) was obtained.

EV recovery was measured based on the amount of ExoSparkler fluorescence labeled with EV. For FCS protein contamination, the protein concentrations in the upper and lower layers of the system to which the FCS sample was added and the BKG system were quantified using a BCA Protein Assay Kit (Thermo Scientific), and the FCS protein concentration was corrected by subtracting the protein concentration of the BKG system from the protein concentration of the FCS sample system.

The FCS protein contamination rate was estimated as the percentage of FCS protein that was incorporated into the lower layer out of the total FCS protein added to the system.

Figure 6 is a plot with two axes representing the EV recovery rate (vertical axis) and the 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.

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.

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 suppressed to a lower level than the PEG-DEX system, and the EV recovery rate was improved to a high level.

The above results show that the concentration method of the present invention, particularly the concentration method of Example 2 including droplet microgelation, has the advantage of not only a higher EV recovery rate in EV concentration, but also a higher ability to remove FCS proteins contained in conditioned media, etc., than the conventional concentration method using ATPS.

Concentration can also 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.

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

Figure 7 shows the results of verification 5, comparing the recovery rate of EVs recovered/concentrated using the concentration method shown in Example 2 with the recovery rate of EVs recovered/concentrated using conventional methods.

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).

In other words, in the concentration method using gelatin-PEG ATPS (0.4% gelatin 100k-7% PEG 8k) according to 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). 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 100 μL.

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 xg, 10 minutes), the upper layer (PEG phase) was separated, and the lower layer (DEX phase) was obtained.

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.

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

For ultracentrifugation, the liquid volume was adjusted to 10 mL with PBS, and then ultracentrifuged (4°C, 200,000 x g, 60 minutes) to obtain a precipitate.

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.

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 showed improved operability and shorter operation time as well as a high EV recovery rate compared to the general concentration method using PEG-DEX ATPS and the polymer precipitation method.

As described above, this embodiment combines particle expulsion due to the excluded volume effect, which is one of the mechanisms of polymer precipitation, with the rapid formation of two phases in an aqueous two-phase system, and the reversible sol/gel conversion properties of the gel-forming polymer, enabling simple and efficient recovery/concentration of biological materials.

That is, by adding a polymer crowder and a gel-forming polymer that can form two phases in combination with the polymer crowder to a liquid sample, it becomes possible to rapidly, easily, and efficiently concentrate biological substances from the liquid sample.

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.

The above describes the aspects of the present invention by illustrating the embodiments, but these are merely examples, 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 (3)

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: 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. The method for concentrating a biological substance according to claim 1, further comprising the step of inducing the separated gel-forming polymer layer to become a sole and melting it.

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