Exosome enrichment by ultrafiltration

Abstract

This invention provides an exosome composition comprising an enriched exosome population. The exosome composition is obtained directly by separation and ultrafiltration, rather than by formulation. This invention also provides a method for producing the exosome composition.

Description

Technical Field

[0001] This invention relates to exosome compositions containing enriched exosomes and their production. Prior Technology

[0002] Exosomes are membrane-bound extracellular vesicles (EVs) produced in the endosome compartments of most eukaryotic cells. Exosomes typically range in size from approximately 40 nm to 160 nm (average approximately 100 nm) in diameter and are of endosome origin. Their surface consists of a lipid bilayer derived from the cell membrane of the donor cell and contains cytosol from the exosome-producing cell, while displaying membrane proteins from the parent cell. Exosomes were initially thought to be a mechanism for removing unwanted proteins. Exosomes can bind to cell surface receptors in a manner similar to intercellular interactions. Furthermore, exosomes can attach to cell membranes and impart new receptors and properties to cells. Therefore, exosomes can fuse with target cells and exchange membrane proteins and cytosol between the two cell types. They are known to mediate intercellular communication and promote protein transfer. It is also known that exosome loading includes a wide range of signaling factors, which are cell-type specific and regulated differently depending on the environment of the secreting cell. As a natural intercellular communication carrier, exosomes have great potential for application in the field of drug delivery due to their natural material delivery characteristics, relatively small molecular structure and excellent biocompatibility. Summary of the Invention

[0003] One aspect of the present invention relates to an exosome composition comprising enriched exosomes having a specific size and molecular weight distribution.

[0004] In one embodiment, the exosome composition comprises an enriched exosome population with a concentration greater than about 1 × 10⁹ exosomes / mL, wherein the exosome composition is obtained directly by separation and ultrafiltration or ultracentrifugation, rather than by formulation.

[0005] In another embodiment, the concentration of exosomes in the composition is greater than about 1 × 10¹⁰ exosomes / mL. In some embodiments, the concentration of exosomes in the composition ranges from about 1 × 10¹⁰ exosomes / mL to about 1 × 10¹⁵ exosomes / mL, about 1 × 10¹⁰ exosomes / mL to about 1 × 10¹⁴ exosomes / mL, about 1 × 10¹⁰ exosomes / mL to about 1 × 10¹³ exosomes / mL, about 1 × 10¹⁰ exosomes / mL to about 1 × 10¹² exosomes / mL, about 1 × 10¹⁰ exosomes / mL to about 1 × 10¹¹ exosomes / mL, or about 1 × 10¹⁰ exosomes / mL to about 5 × 10¹⁰ exosomes / mL.

[0006] In one embodiment, the exosome composition comprises an enriched population of exosomes having an average particle size of about 135 nm to about 150 nm or about 138 nm to about 148 nm.

[0007] In one embodiment, the enriched exosome population described herein comprises exosomes having a molecular weight greater than about 3 kDa, about 10 kDa, about 50 kDa, or about 100 kDa.

[0008] In one embodiment, the enriched exosome population described herein comprises more than 75%, more than 80%, more than 85%, more than 88%, or more than 90% of exosomes having a particle size of less than about 200 nm. In some embodiments, the enriched exosome population described herein comprises more than 75% to 95%, more than 80% to 95%, more than 85% to 95%, more than 75% to 93%, more than 80% to 93%, more than 85% to 93%, or more than 85% to 92% of exosomes having a particle size of less than about 200 nm.

[0009] In one embodiment, the enriched exosome population described herein comprises more than about 10%, more than about 12%, or more than about 15% of exosomes having a particle size of less than 100 nm. In some embodiments, the enriched exosome population described herein comprises about 10% to about 25%, about 10% to about 20%, about 12% to about 25%, about 12% to about 20%, about 15% to about 25%, about 15% to about 20%, about 16% to about 25%, or about 16% to about 20% of exosomes having a size of less than about 100 nm.

[0010] In some embodiments, the enriched exosome populations described herein comprise exosomes with a molecular weight greater than about 3 kDa that have one or more of the following characteristics: Concentration greater than 1 × 10¹⁰ exosomes / mL; Approximately 90% to 93% of exosomes have a particle size of less than approximately 200 nm; Approximately 16% to 20% of the exosomes have a particle size of less than approximately 100 nm; and The average particle size is approximately 135 nm to approximately 145 nm.

[0011] In other embodiments, the concentration is greater than about 2 × 10¹⁰ exosomes / mL or about 3 × 10¹⁰ exosomes / mL (preferably about 3.2 × 10¹⁰ exosomes / mL); about 92% of the exosomes have a particle size of less than about 200 nm; about 17% to about 19% (preferably about 18.6 × 10¹⁰ exosomes / mL) of the exosomes have a particle size of less than about 100 nm; and / or the average particle size is in the range of about 137 nm to about 142 nm (preferably about 140 nm).

[0012] In some embodiments, the enriched exosome populations described herein comprise exosomes with a molecular weight greater than about 10 kDa that have one or more of the following characteristics: Concentration greater than 2 × 10¹⁰ exosomes / mL; Approximately 90% to 93% of exosomes have a particle size of less than approximately 200 nm; Approximately 14% to 18% of exosomes have a particle size of less than approximately 100 nm; and The average particle size is approximately 138 nm to approximately 148 nm.

[0013] In other embodiments, the concentration is greater than about 2.5 × 10¹⁰ exosomes / mL, about 3.0 × 10¹⁰ exosomes / mL, about 3.5 × 10¹⁰ exosomes / mL, or about 4.0 × 10¹⁰ exosomes / mL (preferably about 4.2 × 10¹⁰ exosomes / mL); about 91% of the exosomes have a particle size of less than about 200 nm; about 15% to about 17% (preferably about 16.27 × 10¹⁰ exosomes / mL) of the exosomes have a particle size of less than about 100 nm; and / or the average particle size is in the range of about 140 nm to about 145 nm (preferably about 143.7 nm).

[0014] In some embodiments, the enriched exosome populations described herein comprise exosomes with a molecular weight greater than about 50 kDa that have one or more of the following characteristics: Concentration greater than 1.0 × 10¹⁰ exosomes / mL; Approximately 85% to 90% of exosomes have a particle size of less than approximately 200 nm; Approximately 15% to 25% of the exosomes have a particle size of less than approximately 100 nm; and The average particle size is approximately 140 nm to approximately 150 nm.

[0015] In other embodiments, the concentration is greater than about 1.0 × 10¹⁰ exosomes / mL (preferably about 1.44 × 10¹⁰ exosomes / mL); about 88.86% of the exosomes have a particle size of less than about 200 nm; about 18% to about 20% (preferably about 19.21 × 10¹⁰ exosomes / mL) of the exosomes have a particle size of less than about 100 nm; and / or the average particle size is about 142 nm to 148 nm (preferably about 145 nm).

[0016] In some embodiments, the enriched exosome populations described herein comprise exosomes with a molecular weight greater than about 100 kDa that have one or more of the following characteristics: Concentration greater than 1.0 × 10¹⁰ exosomes / mL; Approximately 88% to 92% of exosomes have a particle size of less than approximately 200 nm; Approximately 15% to 20% of the exosomes have a particle size of less than approximately 100 nm; and The average particle size is approximately 138 nm to approximately 145 nm.

[0017] In other embodiments, the concentration is greater than about 1.2 × 10¹⁰ exosomes / mL (preferably about 1.48 × 10¹⁰ exosomes / mL); about 90.93% of the exosomes have a particle size of less than about 200 nm; about 16% to about 19% (preferably about 18.7 × 10¹⁰ exosomes / mL) of the exosomes have a particle size of less than about 100 nm; and / or the average particle size is in the range of about 140 nm to about 144 nm (preferably about 142.2 nm).

[0018] In one embodiment, the exosomes described herein are derived from stem cells. In some embodiments, the stem cells are embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), hematopoietic stem cell transplantation (HSCT), or induced pluripotent stem cells (iPS cells or iPSCs).

[0019] Another aspect of the present invention relates to a method for generating such exosome populations.

[0020] In one embodiment, the present invention provides a method for producing the exosome composition described herein, comprising the following steps: Provide the required amount of cells in the culture medium; The culture medium was centrifuged to remove cell debris, and the resulting supernatant was then filtered to remove apoptotic cells and small cells. The resulting supernatant was subjected to ultrafiltration using a membrane with a molecular weight cutoff of approximately 3 kDa to approximately 100 kDa; and Polymer-based precipitation is used to precipitate exosomes to obtain the exosome composition containing enriched exosomes.

[0021] In one embodiment, the cells used in the method are stem cells. In some embodiments, the stem cells are embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), hematopoietic stem cell transplantation (HSCT), or induced pluripotent stem cells (iPS cells or iPSCs).

[0022] In some embodiments of the present invention, ultrafiltration is performed via a concentrator rotating column using a membrane with a molecular weight cutoff.

[0023] In one embodiment, the present invention provides a method for producing the exosome composition described herein, comprising the following steps: Provide the required amount of cells in the culture medium; The culture medium was centrifuged to remove cell debris, and the resulting supernatant was then filtered to remove apoptotic cells and small cells; and The resulting supernatant was ultracentrifuged at about 80,000 g to about 12,000 g for about 50 minutes to about 90 minutes to obtain the exosome composition containing enriched exosomes.

[0024] In one embodiment, the ultracentrifugation method ranges from about 80,000 g to about 12,000 g, about 85,000 g to about 11,500 g, about 90,000 g to about 11,000 g, or about 95,000 g to about 10,500 g, for about 50 minutes to about 90 minutes, about 55 minutes to about 85 minutes, about 60 minutes to about 80 minutes, or about 65 minutes to about 75 minutes. Simple Explanation of the Diagram

[0025] Figure 1 shows the time consumption results of ultrafiltration.

[0026] Figure 2 shows the results of nanoparticle tracking analysis in Example 1.

[0027] Figure 3 shows the parameters of the nanoparticle tracking analysis. The average size, D90, and D10 are shown in the 3 kDa, 10 kDa, 50 kDa, and 100 kDa ultrafiltration methods.

[0028] Figure 4 shows the performance of exosome markers. Alix is ​​a protein within the exosome. CD81 is a surface protein of the exosome, which is reduced at 50 kDa and 100 kDa compared to 3 kDa.

[0029] Figure 5 illustrates the particle concentration performance. The particle concentration performance was estimated. Nanoparticle tracking analysis showed a decrease in particle concentration at 50 kDa and 100 kDa compared to 3 kDa.

[0030] Figure 6 shows the penetration electron microscopy (TEM) analysis of UCMSC-derived exosomes.

[0031] Figure 7 shows the results of nanoparticle tracking analysis in Examples 1 and 3.

[0032] Figure 8 shows the parameters of nanoparticle tracking analysis in Examples 1 and 3.

[0033] Figure 9 shows the particle concentration and exosome labeling in Examples 1 and 3. Implementation

[0034] Unless the context otherwise specifies or indicates, the terms "a / an" and "the" mean "one or more".

[0035] As used herein, “approximately” will be understood by those generally familiar with the technology and will vary to some extent depending on the context in which it is used. If the use of a term is unclear to those generally familiar with the technology in the context in which it is used, then “approximately” will mean that the specific term is added to or subtracted by 10%.

[0036] As used herein, the term "exosome" refers to any extracellular vesicle derived from any bodily fluid (e.g., blood) of a human or animal, including but not limited to autologous exosomes, universal donor exosomes, allogeneic exosomes, and modified exosomes.

[0037] The term "formulation" refers to a preparation in which the biological activity of the active agent is permitted to be effective.

[0038] As used herein, the term "stem cell" refers to cells in an undifferentiated or partially differentiated state that possess self-renewal properties and the developmental potential to naturally differentiate into more differentiated cell types. This developmental potential has no specific implied meaning (i.e., totipotency, pluripotency, etc.). Self-renewal means that stem cells can proliferate and produce more of these stem cells while maintaining their developmental potential. Therefore, the term "stem cell" refers to any subpopulation of cells that, under certain conditions, possess the developmental potential to differentiate into a more specific or differentiated phenotype, and in some cases retain the ability to proliferate without substantially differentiating.

[0039] As used in this article, the term "derived from" should be understood as indicating that a particular sample or sample group is derived from a specified species, but not necessarily directly from a specified source.

[0040] Exosomes, either directly or as a delivery medium for drug payloads, are being actively explored as therapeutic agents. Compared to liposomes, injected exosomes are more efficient at entering other cells, can deliver functional payloads with minimal immune clearance, and exhibit good tolerability upon exogenous administration.

[0041] Exosome heterogeneity can reflect its size, contents, functional effects on recipient cells, and cellular origin. Size variability can be attributed to the inhomogeneous infolding of the boundary membrane of multivesicular bodies (MVBs), leading to differences in the total fluid and solid content, or to other EV separation methods. Fine fractionation methods involving EVs demonstrate that exosomes can contain subpopulations defined by different size ranges. Size heterogeneity can also lead to varying amounts of exosome contents. The cellular microenvironment and inherent biology can influence exosome contents and their biomarkers (Raghu Kalluri and Valerie S. LeBleu, Science Vol. 367, No. 6478, February 7, 2020).

[0042] This article reveals exosomes comprising enriched exosome populations at concentrations greater than approximately 1 × 10⁹ exosomes / mL. Methods for generating the revealed exosomes are also considered.

[0043] Exosome compositions can be derived from any number of tissue sources, such as muscle, fat, organs, or bone or bone marrow. Various cells can be used to prepare the exosome compositions of this invention. Specifically, the exosome compositions can be derived from cells. In some embodiments of this invention, the cells are derived from eukaryotic organisms or cell lines. In some embodiments of this invention, the cells are derived from plants or animals. The cells may be genetically modified. In some embodiments of this invention, the cells are stem cells. In some embodiments, the stem cells are embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), hematopoietic stem cell transplantation (HSCT), or induced pluripotent stem cells (iPS cells or iPSCs).

[0044] Specific methods are used to produce the exosome compositions of the present invention. In the methods for producing the exosome compositions of the present invention, a desired amount of cells is first provided. In some embodiments of the invention, the cells are cultured in a culture medium for a period of time sufficient to obtain the desired amount of cells. In some embodiments of the invention, the cell quantity is about 1 × 10⁵ cells / mL to about 1 × 10⁸ cells / mL; about 2 × 10⁵ cells / mL to about 8 × 10⁷ cells / mL; about 4 × 10⁵ cells / mL to about 6 × 10⁷ cells / mL; about 6 × 10⁵ cells / mL to about 4 × 10⁷ cells / mL; about 8 × 10⁵ cells / mL to about 2 × 10⁷ cells / mL; about 1 × 10⁶ cells / mL to about 1 × 10⁷ cells / mL; about 2 × 10⁶ cells / mL to about 8 × 10⁶ cells / mL; or about 4 × 10⁶ cells / mL to about 6 × 10⁶ cells / mL. The culture method depends on the cells. In some embodiments of the present invention, the culture medium is a conditioned medium for obtaining a desired amount of cells with specific characteristics. For example, a conditioned medium is provided to maintain cells in an undifferentiated or differentiated stage.

[0045] The culture medium containing the required amount of cells undergoes a pre-cleaning procedure to remove dead cells and / or cell debris. In one embodiment of the invention, the culture medium is centrifuged to remove dead cells. In some embodiments of the invention, dead cells may be removed at about 300 g, about 350 g, or about 400 g for about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, or about 15 minutes. In one embodiment of the invention, the culture medium is centrifuged to remove cell debris. In some embodiments of the present invention, cell debris may be removed in quantities of about 1500 g, about 1600 g, about 1700 g, about 1700 g, about 1800 g, about 1900 g, about 2000 g, about 2100 g, about 2200 g, about 2300 g, about 2400 g, or about 2500 g for about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes.

[0046] The supernatant obtained after filtering to remove dead cells and / or cell debris is then used to remove apoptotic cells and small cells. In one embodiment of the invention, the obtained supernatant is filtered through a filter of about 0.15 μm, about 0.16 μm, about 0.18 μm, about 0.20 μm, about 0.22 μm, about 0.24 μm, about 0.26 μm, or about 0.28 μm.

[0047] In one embodiment of the invention, the supernatant obtained after pre-cleaning is then subjected to ultrafiltration using a membrane having a molecular weight cutoff of about 3 kDa to about 100 kDa. In some embodiments of the invention, the ultrafiltration is performed via a concentrator rotating column. In some embodiments of the invention, the membrane has a molecular weight cutoff of about 3 kDa, and the ultrafiltration is performed for 480 minutes. In some embodiments of the invention, the membrane has a molecular weight cutoff of about 10 kDa, and the ultrafiltration is performed for 60 minutes.

[0048] One type of membrane filtration involves forces (such as pressure or concentration gradient) that cause separation via a semi-permeable membrane. Ultrafiltration membranes are typically characterized by their molecular weight cutoff. Suspended solids and higher molecular weight solutes are retained in the retention, while water and lower molecular weight solutes pass through the membrane in the permeate. Different types of modules can be used in ultrafiltration processes. Examples of such modules include tubular elements using polymeric membranes cast on the inside of plastic or paper tubes; hollow fiber designs containing multiple hollow fibers; spirally wound modules in which flat membrane sheets are separated by a thin mesh spacer material rolled around a central perforated tube and mounted into a tubular steel pressure vessel shell; and plate and frame assemblies using membranes placed on a plate and separated by a mesh material through which the filtrate passes.

[0049] The supernatant obtained after ultrafiltration is then subjected to exosome precipitation for enrichment. In some embodiments of the invention, the supernatant obtained after ultrafiltration is precipitated using a polymer-based precipitant. The resulting supernatant contains an enriched exosome population with a concentration greater than about 1 × 10⁹ exosomes / mL.

[0050] Cell conditioned medium is a key step in exosome extraction. Large quantities of cell conditioned medium lead to increased costs in exosome extraction and enrichment. Ultrafiltration is used to concentrate the cell conditioned medium for exosome enrichment. This invention provides an efficient manufacturing method for the enrichment of exosomes from extraction.

[0051] In one embodiment of the invention, the supernatant obtained after pre-cleaning is then subjected to ultracentrifugation at a rate of about 80,000 g to about 12,000 g for about 50 minutes to about 90 minutes.

[0052] Exosome compositions can be identified by determining their molecular weight, average particle size, exosome concentration, and particle size range. The results are described herein. In some embodiments of the invention, in nanoparticle tracking analysis, exosome extraction following 3 kDa ultrafiltration showed an increase in particle number compared to conventional ultracentrifugation. Exosome labeling performance was also increased in 3 kDa ultrafiltration. Furthermore, exosome extraction efficiency following 10 kDa ultrafiltration was similar to that of ultracentrifugation. The average size was approximately 150 nm to approximately 160 nm, and the size distribution was approximately 100 nm to approximately 200 nm.

[0053] The exosome compositions of this invention can be used to treat any of the following diseases, including but not limited to: cancer and neoplasms; infectious diseases; cardiovascular diseases; diabetes, including type 1 and type 2 diabetes; liver diseases; obesity; rare diseases; gastrointestinal diseases; bone diseases; and sickle cell diseases. The exosome compositions of this invention can also be used as cell therapy agents; carriers and cell engineering modifications; and for pharmacological and toxicological analysis and development.

[0054] For illustrative purposes only, specific embodiments have been used to describe the invention herein. However, it will be apparent to those skilled in the art that the principles of the invention can be embodied in other ways. Therefore, the invention should not be construed as being limited to the specific embodiments and the scope of the claims. [Example] []

[0055] [Example] [1] [Production of Exosome Compositions] []

[0056] Umbilical cord mesenchymal stem cells (MSCs) were cultured in conditioned medium to reach a cell count of 1.5 × 10⁶ cells / mL. The medium was centrifuged at 350 g for 10 minutes at 4°C to remove dead cells. The resulting supernatant was further centrifuged at 2,000 g for 15 minutes at 4°C to remove cell debris. The supernatant was filtered through a 0.22 μm filter to remove apoptotic cells and small cells.

[0057] 30 ml of pre-cleaned cell conditioned medium was ultrafiltered via VIVASPIN® 6 (sample loading: 2 mL to 6 mL) and 20 (sample loading: 5 mL to 20 mL) rotary columns of 3 kDa, 10 kDa, 50 kDa, and 100 kDa. The pore size of the membrane in the rotary column was proportional to kilodaltons. The time required to concentrate 30 mL of pre-cleaned cell conditioned medium to 1 mL was estimated and is shown in Table 1 and Figure 1. The results showed that the 3 kDa ultrafiltration method (480 min) took longer than the 10 kDa (60 min), 50 kDa (55 min), and 100 kDa (50 min) methods. Table 1 Ultrafiltration (30 mL to mL) 3 kDa 10 kDa 50 kDa 100 kDa aperture 1.2 nm 2.5 nm 7 nm 10 nm Time (minutes) 480 minutes 60 minutes 55 minutes 50 minutes

[0058] Exosomes were precipitated using the TOOLSharp® Cell Culture Media Exosome Extraction Kit based on polymer precipitation. An exosome composition containing enriched exosome populations was subsequently obtained. The exosome composition was identified as having the characteristics shown in Table 2 and Figures 2 and 3. The size distribution of the exosomes was analyzed using nanoparticle tracking analysis. As shown in Figure 2, the size distributions at 3 kDa, 10 kDa, 50 kDa, and 100 kDa were similar and between 100 nm and 200 nm. As shown in Figure 3, the average size was approximately 150 nm to 160 nm. D90 and D10 showed a size distribution between 100 nm and 200 nm. Table 2 [3 kDa] [10 kDa] [50 kDa] [100 kDa] [CCM] [volume] 30 ml 30 ml 30 ml 30 ml [average value(] [size)] 139.7 nm 143.7 nm 145.0 nm 142.2 nm [Exosome concentration] 3.20×10 10 4.26×10 10 1.44×10 10 1.48×10 10 [D10] 91.9 nm 94.9 nm 92.6 nm 91.3 nm [D90] 191.8 nm 197.8 nm 205.9 nm 197.5 nm [<200 nm] 91.90% 90.71% 88.86% 90.93% [<100 nm] 18.66% 16.27% 19.21% 18.70%

[0059] [Example] [2] [Exosome composition containing exosome populations] []

[0060] The amounts of Alix and CD81 on UCMSC-derived exosomes obtained in Example 1 were measured by electrophoresis, and are shown in Figure 4. Compared with 3 kDa, Alix and CD81 decreased at 50 kDa and 100 kDa.

[0061] Nanoparticle tracking analysis showed that particle concentrations decreased at 50 kDa and 100 kDa compared to 3 kDa.

[0062] The penetrating electron microscopy (TEM) analysis of UCMSC-derived exosomes is shown in Figure 6.

[0063] [Example] [3] [Production of Exosome Compositions] [-] [Ultracentrifugation] []

[0064] Umbilical cord mesenchymal stem cells (MSCs) were cultured in conditioned medium to reach a cell count of 1.5 × 10⁶ cells / mL. The medium was centrifuged at 350 g for 10 minutes at 4°C to remove dead cells. The resulting supernatant was further centrifuged at 2,000 g for 15 minutes at 4°C to remove cell debris. The supernatant was filtered through a 0.22 μm filter to remove apoptotic cells and small cells.

[0065] 12 mL of pre-cleaned cell conditioned medium was ultracentrifuged at 100,000 g for 70 min. The size distribution of exosomes in this example (ultracentrifugation) and Example 1 (3 kDa and 10 kDa) was analyzed by nanoparticle tracking analysis, and is shown in Figure 7. The results show a similar size distribution between 100 nm and 200 nm.

[0066] The parameters of nanoparticle tracking analysis in this example (ultracentrifugation) and Example 1 (3 kDa and 10 kDa) were analyzed by nanoparticle tracking analysis, and are shown in Figure 8. The average size of all particles is approximately 150 nm to 160 nm. D90 and D10 show the size distribution between 100 nm and 200 nm.

[0067] The particle concentrations and the amounts of Alix and CD81 on UCMSC-derived exosomes obtained in this example (ultracentrifugation) and Example 1 (3 kDa and 10 kDa) were measured and are shown in Figure 9. The particle concentration was increased in the 3 kDa ultrafiltration method compared to the 10 kDa and ultracentrifugation methods. The levels of Alix and CD81 also increased in the 3 kDa method.

Claims

1. An exosome composition comprising an enriched exosome population at a concentration greater than about 1 × 10⁹ exosomes / mL, wherein the exosome composition is obtained directly by ultrafiltration rather than by formulation; wherein the enriched exosome population comprises exosomes with a molecular weight greater than about 10 kDa, having the following characteristics: a concentration greater than about 2 × 10¹⁰ exosomes / mL; 90% to 93% of the exosomes having a mass division diameter less than about 200 nm; 14% to 18% of the exosomes having a mass division diameter less than about 100 nm; and an average mass division diameter of 138 nm to 148 nm; wherein the ultrafiltration is performed on the resulting supernatant using a membrane with a molecular weight cutoff of 10 kDa for less than 60 minutes; and wherein the exosomes are mesenchymal stem cell (MSC) exosomes.

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