Method for lyophilizing exosome

Abstract

A method of lyophilizing exosomes using a cryoprotectant comprising methionine, mannitol and trehalose is disclosed. The lyophilized exosome product shows a good appearance which maintains a porous sponge shape without forming ice crystals. In addition, the lyophilized exosome product can be applied to a pharmaceutical composition, a skin external preparation and a cosmetic composition. For example, the lyophilized exosome product can be used as a solution obtained by simply mixing it with a diluent.

Claims

1. A composition for lyophilizing exosomes comprising: an aqueous solution prepared by adding a cryoprotectant to an aqueous solution containing ascorbic acid and retinol, wherein the cryoprotectant comprises methionine, mannitol and trehalose; and exosomes.

2. A method for lyophilizing exosomes, the method comprising the following steps of: (a) providing exosomes; (b) treating the exosomes with a cryoprotectant comprising methionine, mannitol, trehalose, ascorbic acid and retinol; and (c) lyophilizing the exosomes treated with the cryoprotectant; wherein step (b) comprises mixing the exosomes with the cryoprotectant.

3. A method for lyophilizing exosomes, the method comprising the following steps of: (a) providing exosomes; (b) treating the exosomes with a cryoprotectant comprising methionine, mannitol, trehalose, ascorbic acid and retinol; and (c) lyophilizing the exosomes treated with the cryoprotectant; wherein step (c) sequentially comprises: freezing under atmospheric pressure at −50° C. for 10 hours to 15 hours; first drying under vacuum at −50° C. for 50 hours to 60 hours; second drying under vacuum at −20° C. for 1 hour to 3 hours; and third drying under vacuum at 10° C. for 30 minutes to 2 hours.

4. A lyophilized formulation of exosomes comprising as active ingredients, exosomes; and methionine, mannitol, trehalose, ascorbic acid and retinol.

5. A composition comprising the lyophilized formulation according to claim 4.

6. The composition of claim 5, wherein the composition is a pharmaceutical composition prepared as an injectable formulation.

7. The composition of claim 5, wherein the composition is a cosmetic composition.

8. The composition of claim 5, wherein the composition is a skin external preparation.

9. A lyophilized formulation of exosomes comprising as active ingredients, exosomes; and methionine, mannitol, trehalose, ascorbic acid and retinol; wherein the lyophilized formulation is used as a solution obtained by mixing the lyophilized formulation with a diluent; wherein the diluent is water for injection, physiological saline, phosphate buffered saline, purified water, or deionized water; and wherein the diluent further comprises hyaluronic acid or hyaluronate.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIGS. 1A to 1E show the results of analyzing the physical properties of exosomes obtained according to one embodiment of the present invention. “FIG. 1A” shows the particle size distribution and the number of particles obtained by nanoparticle tracking analysis (NTA). “FIG. 1B” shows particle images obtained by transmitted electron microscopy (TEM)”. “FIG. 1C” shows the results of Western blot analysis for positive markers of exosomes obtained according to one embodiment of the present invention. “FIG. 1D” shows the results of Western blot analysis for negative markers of exosomes obtained according to one embodiment of the present invention. “FIG. 1E” shows the results of flow cytometry of CD9, CD63 and CD81 in the analysis of markers for exosomes obtained according to one embodiment of the present invention.

(2) FIG. 2 depicts a photograph showing a good appearance of exosomes lyophilized according to one embodiment of the present invention.

(3) FIGS. 3A to 3G are photographs each of which shows, after performing lyophilization using different combinations of cryoprotectant components, the appearance of lyophilized exosomes obtained according to each of the combinations of cryoprotectant components.

(4) FIGS. 4 to 7 show the results of analyzing inflammatory cytokines using a multiplex panel. In FIGS. 4 to 7, when RAW 264.7 cells were treated with different concentrations (expressed as the number of particles per mL) of stem cell-derived exosomes (exosomes isolated and purified from conditioned media of stem cells) prepared in Example 2, and then treated with LPS, the LPS-induced production of IL-1β, IL-6, IL-27 and IFN-β in the cells decreased in a manner of depending on the concentration of the exosomes.

(5) FIGS. 8 to 11 show the results of analyzing inflammatory cytokines using a multiplex panel. In FIGS. 8 to 11, when RAW 264.7 cells were treated with different concentrations (expressed as the number of particles per mL) of aqueous solutions obtained and diluted by mixing a lyophilized formulation of stem cell-derived exosomes (prepared in Example 4-1) with a culture medium, and then treated with LPS, the LPS-induced production of IL-1β, IL-6, IL-27 and IFN-β in the cells remarkably decreased in a manner of depending on the concentration of the lyophilized formulation.

EXAMPLES

(6) Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only to illustrate the present invention and are not intended to limit or restrict the scope of the present invention. Those that can be easily inferred by those skilled in the art from the detailed description and examples of the present invention are interpreted as falling within the scope of the present invention. References referred to in the present invention are incorporated herein by reference.

(7) Throughout the present specification, it is to be understood that, when any part is referred to as “comprising” any component, it does not exclude other components, but may further include other components, unless otherwise specified.

Example 1: Cell Culture

(8) According to a cell culture method known in the technical field to which the present invention pertains, adipose-derived stem cells were cultured at 37° C. under 5% CO.sub.2. Next, the cells were washed with phosphate-buffered saline (purchased from ThermoFisher Scientific), and then the medium was replaced with serum-free, phenol red-free medium, and the cells were cultured for 1 to 10 days. The supernatant (hereinafter, referred to as “conditioned medium”) was recovered.

(9) In order to obtain exosomes having a uniform particle size distribution and high purity in an exosome isolation process, 2 wt % of trehalose was added to the conditioned medium. After addition of trehalose, the conditioned medium was filtered through 0.22 μm filter to remove impurities, such as cell debris, waste, macroparticles and the like. From the filtered conditioned medium, exosomes were immediately isolated. In addition, the filtered conditioned medium was stored in a refrigerator (10° C. or below), and then used for exosome isolation. Furthermore, the filtered conditioned medium was freeze-stored in an ultra-low temperature freezer at −60° C. or below, thawed, and then subjected to exosome isolation. Thereafter, exosomes were isolated from the conditioned medium by Tangential Flow Filtration (TFF).

Example 2: Isolation and Purification of Exosomes by TFF Method

(10) For isolating, concentrating and diafiltrating exosomes from the conditioned medium filtered through 0.22 μm filter in Example 1, TFF method was used. As a filter for TFF method, a cartridge filter (known as a hollow fiber filter; purchased from GE Healthcare) or a cassette filter (purchased from Pall, Sartorius or Merck Millipore) was used. The TFF filter may be selected with various molecular weight cutoffs (MWCOs). Using the filter having selected MWCO, exosomes were isolated and concentrated, and particles, proteins, lipids, nucleic acids, low-molecular-weight compounds, etc., were removed, which are smaller than the MWCO.

(11) To isolate and concentrate exosomes, a TFF filter having MWCO of 100,000 Da (Dalton), 300,000 Da or 500,000 Da was used. Exosomes were isolated from the conditioned medium by removing substances smaller than the MWCO and concentrating the conditioned medium to a volume of about 1/100 to 1/25 by the TFF method.

(12) The isolated and concentrated solution of exosomes was additionally subjected to diafiltration. The diafiltration was performed continuously (continuous diafiltration) or discontinuously (discontinuous diafiltration), using a buffer having at least 4 times, preferably at least 6 to 10 times, more preferably at least 12 times volume of the starting volume. To obtain exosomes having a uniform particle size distribution and high purity, 2 wt % trehalose in PBS was added to the buffer.

Example 3: Analysis of Characteristics of Isolated Exosomes

(13) The particle size and concentration of the isolated exosomes were measured by nanoparticle tracking analysis (NTA) instrument (purchased from Malvern). The uniformity and size of the isolated exosomes were analyzed by transmission electron microscopy (TEM). FIGS. 1A and 1B show the results of NTA and TEM of the exosomes isolated by the isolation method according to one embodiment of the present invention.

(14) FIG. 1C shows the results of Western blot analysis for positive markers of the exosomes isolated by the isolation method according to one embodiment of the present invention. As shown therein, the presence of CD63, CD9, CD81, Alix and TSG101 markers was confirmed. As antibodies for each of the markers, anti-CD63, anti-CD9, anti-CD81, anti-Alix and anti-TSG101 were used, respectively.

(15) FIG. 1D shows the results of Western blot analysis for negative markers of the exosomes isolated by the isolation method according to one embodiment of the present invention. As antibodies for each of the markers, anti-GM130 and anti-Calnexin were used, respectively. GM130 and Calnexin are negative markers that should not be present in exosomes when the characteristics of the exosomes are analyzed. As shown in FIG. 1D, it was confirmed that GM130 and Calnexin were present in a lysate in adipose-derived stem cells, but were not present in the exosomes isolated by the isolation method according to one embodiment of the present invention. Therefore, when considering the results shown in FIGS. 1C and 1D together, it can be seen that the exosomes isolated by the isolation method according to one embodiment of the present invention are exosomes satisfying the characteristics of the positive markers and negative markers.

(16) FIG. 1E shows the results of flow cytometry of the exosomes isolated by the isolation method according to one embodiment of the present invention. As shown therein, the presence of CD9, CD63 and CD81 markers was confirmed. To isolate CD81-positive exosomes, an Exosome-Human CD81 Isolation/Detection Reagent kit (purchased from ThermoFisher Scientific) was used according to the manufacturer's instruction. The markers were stained with PE-Mouse anti-human CD9 (purchased from BD), PE-Mouse anti-human CD63 (purchased from BD) or PE-Mouse anti-human CD81 (purchased from BD), and then analyzed using a flow cytometer (ACEA Biosciences).

(17) Meanwhile, it is to be understood that the exosomes that are used in the lyophilizing method of the present invention are not limited to the exosomes of the Examples as described above, it is possible to use various exosomes that are being used in the art or can be used in the future. In addition, it should be noted that the exosomes isolated according to the above Examples should be understood as an example of exosomes that may be used in the lyophilizing method of the present invention, and the scope of the present invention is not limited thereto.

Example 4: Lyophilization of Exosomes

Example 4-1: Lyophilization Conditions

(18) For lyophilization of exosomes, a cryoprotectant comprising methionine, mannitol and trehalose was prepared. An aqueous solution was prepared by adding the cryoprotectant was added to 1 mL of an aqueous solution containing 0.5 mg/mL each of ascorbic acid and retinol (prepared by BIO-FD&C Co., Ltd., Hwasun-gun, Jeollanam-do, Korea). Although the cryoprotectant was added to the solution containing ascorbic acid and retinol in this Example, an aqueous solution may also be prepared by adding the cryoprotectant to water for injection, purified water, physical saline, or deionized water. The concentration of each of methionine, mannitol and trehalose in the aqueous solution was adjusted to 9 mg/mL.

(19) The exosomes (5×10.sup.8 particles/mL) prepared in Example 2 were mixed with the aqueous solution containing the cryoprotectant, and then lyophilized using a lyophilization system (manufactured by VIRTIS, ITEM No.: 344424) under the conditions shown in Table 1 below. The lyophilization was performed in the order of conditions 1, 2, 3, 4, 5, 6, 7 and 8 as shown in Table 1 below.

(20) TABLE-US-00001 TABLE 1 Lyophilization conditions Total time (min) 4320 Conditions Time (min) Temperature (° C.) Pressure (mmHg) 1 700 −50 760 2 60 −50 760 3 999 −50 0 4 999 −50 0 5 999 −50 0 6 370 −50 0 7 120 −20 0 8 73 10 0

(21) After the exosomes were treated with the cryoprotectant comprising methionine, mannitol and trehalose, and lyophilized, the appearance thereof was examined. As a result, it can be seen that the exosomes were milky white in color and showed a good appearance which maintains a porous sponge shape (FIG. 2). That is, the method for lyophilizing exosomes according to the present invention is able to produce a lyophilized product having a good appearance by prolonging the drying time under vacuum and using the cryoprotectant having the combination of methionine, mannitol and trehalose.

Example 4-2: Comparison of Appearances of Lyophilized Exosomes Depending on Cryoprotectant Components

(22) Meanwhile, the appearances of exosomes lyophilized using various cryoprotectants comprising at least one of methionine, mannitol and trehalose (hereinafter, referred to as cryoprotectant components) were compared. According to the method described in Example 4-1 above, seven different aqueous solutions were prepared by adding the cryoprotectant components alone, combinations of two components, or a combination of three components. The concentration of each of the cryoprotectant components in each of the aqueous solutions was adjusted to 9 mg/mL. According to the lyophilization conditions and method described in Example 4-1 above, the exosomes (5×10.sup.8 particles) prepared in Example 2 above were mixed with the respective aqueous solution containing each of the cryoprotectant components alone, each of the combinations of two components, or the combination of three components, and then lyophilized.

(23) The external appearances of the lyophilized exosome products were photographed and evaluated (FIGS. 3A to 3G). According to the states of the cake shapes of the lyophilized exosome products, the appearances of the lyophilized exosome products were ranked and relatively evaluated in a 5-point scale ranging from 1 (the worst cake appearance) to 5 (the best cake appearance). Table 2 below shows the results of evaluating the appearances of the lyophilized exosome products according to the combinations of the cryoprotectant components.

(24) TABLE-US-00002 TABLE 2 Comparison of appearances of lyophilized exosome products according to combinations of cryoprotectant components Composition of cryoprotectant components Methionine + mannitol + trehalose Methionine + Methionine + Trehalose + (present Mannitol Trehalose Methionine trehalose mannitol mannitol invention) Evaluated 1 1 4 3 4 2 5 score FIGS. FIG. 3A FIG. 3B FIG. 3C FIG. 3D FIG. 3E FIG. 3F FIG. 3G

(25) As shown in FIGS. 3A to 3G and Table 2 above, it can be seen that the product obtained by lyophilizing exosomes using the cryoprotectant having the combination of methionine, mannitol and trehalose has the best external appearance, however, the external appearances of the products obtained by lyophilizing exosomes using the one component or the combinations of the two components are poorer than that of the product of the present invention.

Example 5: Evaluation of Effect of Decreasing Inflammatory Cytokine Production

(26) The effects of stem cell-derived exosomes (Example 2) and the lyophilized formulation (Example 4-1) of stem cell-derived exosomes upon decreases in inflammatory cytokine production in mouse macrophage RAW 264.7 cells were evaluated as follows.

(27) RAW 264.7 cells were suspended in DMEM (Dulbecco Modified Eagle Medium; purchased from ThermoFisher Scientific) containing 10% FBS (Fetal Bovine Serum) and 1% penicillin-streptomycin, and then seeded into a 96-well plate at a density of 2.5×10.sup.4 cells/well. Next, the cells were treated with difference concentrations (expressed as the number of particles per mL) of stem cell-derived exosomes (exosomes isolated and purified from conditioned media of stem cells) prepared in Example 2, and then cultured in an incubator at 37° C. under 5% CO.sub.2 for 24 hours. In addition, in the same manner as the above-described method of treatment with stem cell-derived exosomes, RAW 264.7 cells were treated with aqueous solutions obtained and diluted by mixing the lyophilized formulation of stem cell-derived exosomes (5×10.sup.9 particles/vial) of Example 4-1 with a culture medium, and then the treated RAW 264.7 cells were cultured in an incubator at 37° C. under 5% CO.sub.2 for 24 hours. Meanwhile, as a positive control, dexamethasone was used (indicated as DEX in FIGS. 4 to 11).

(28) Thereafter, the RAW 264.7 cells were treated with 100 ng/mL of LPS (purchased from Sigma), and cultured in an incubator at 37° C. under 5% CO.sub.2 for 24 hours, thus inducing activation of the cells.

(29) After completion of the culture, the culture supernatant of the RAW 264.7 cells was collected, and the production of IL-1β, IL-6, IL-27 and IFN-β in the culture supernatant was measured using a mouse inflammation panel for LEGENDplex™ bead-based immunoassay (purchased from Biolegend) and NovoCyte Flow Cytometer (purchased from ACEA) in order to evaluate anti-inflammatory effects.

(30) In addition, an MTT assay was performed to measure the change in cell viability caused by stem cell-derived exosomes, and the lyophilized formulation of stem cell-derived exosomes of the present invention, respectively, and to normalize the cytokine production therethrough. After the completion of culture, the culture medium of the RAW 264.7 cells was replaced with a DMEM medium containing 0.5 mg/mL of thiazolyl blue tetrazolium bromide (purchased from Sigma) and cultured for 1 hour. Next, the supernatant was removed in such a manner that the formazan formed at the bottom of the cell culture plate was not scattered. Subsequently, the formazan was dissolved by dimethyl sulfoxide (purchased from AMRESCO), and the absorbance was measured at 570 nm to determine the cell viability. In addition, the production of each of cytokines (IL-1β, IL-6, IL-27 and IFN-β) was normalized by the cell viability.

(31) As shown in FIGS. 4 to 7, from the results of analyzing the inflammatory cytokines using the multiplex panel, it can be seen that when the RAW 264.7 cells were treated with the stem cell-derived exosomes before treating the cells with LPS, the LPS-induced production of each of IL-1β, IL-6, IL-27 and IFN-β decreased in a manner of depending on the concentration of the exosomes. In addition, as shown in FIGS. 8 to 11, from the results of analyzing the inflammatory cytokines using the multiplex panel, it can be seen that when the RAW 264.7 cells were treated with the lyophilized formulation of stem cell-derived exosomes before treating the cells with LPS, the LPS-induced production of each of IL-1β, IL-6, IL-27 and IFN-β remarkably decreased in a manner of depending on the concentration of the lyophilized formulation.

(32) From these results, it can be seen that the lyophilized formulation of stem cell-derived exosomes according to the present invention is able to stably maintain the anti-inflammatory efficacy of stem cell-derived exosomes contained therein as an active ingredient, and thus there is no possible change in the physical properties of exosomes during lyophilization, storage and distribution. Therefore, the lyophilized formulation of stem cell-derived exosomes according to the present invention is able to stabilize exosomes and make them commercially useful.

(33) Although the present invention has been described with reference to the embodiments, the scope of the present invention is not limited to these embodiments. Any person skilled in the art will appreciate that various modifications and changes are possible without departing from the spirit and scope of the present invention and these modifications and changes also fall within the scope of the present invention.