Patent Application
20240043797
This application claims priority to and the benefit of Korean Application Number 10-2020-0174891, filed in the Korean Intellectual Property Office on Dec. 14, 2020, the entire content of which is incorporated herein by reference.
The present disclosure relates to a method for mass production of extracellular vesicles, using a peptide derived from Noxa protein and mesenchymal stem cells and, specifically, a method for mass production of extracellular vesicles, wherein mesenchymal stem cells are cultured in a medium composition containing a Noxa protein-derived peptide, glucose, sucrose, and 3-(N-morpholino)propanesulfonic acid (MOPS), whereby extracellular vesicles having wound healing and immunomodulatory effects can be obtained with high purity at high yield.
Extracellular vesicles are lipid bilayer-structured vesicles with various sizes, secreted from various eukaryotic cells, such as insects, plants, and microorganisms as well as humans and animals. Of them, vesicles in nano-sizes are called exosomes.
Exosomes retain specific molecules such as proteins, nucleic acids, lipids, and carbohydrates that are contained in cells, stably protect the specific molecules with the lipid bilayer, and play a role in transmitting information to other cells after being released.
Exosomes are attracting attention as a new drug delivery vehicle. Exosomes not only enter cells more easily than liposomes, but also encounter little resistance from the immune system. In addition, an abundant amount of ligands present on the membrane surface of exosomes exhibits the possibility of cell-specific delivery through receptors.
In relation to a regenerative medicine or immune disease treatment using stem cells, a therapy using stem cell-derived extracellular vesicles, which replaces a cell therapy designed to transplant live stem cells into a lesion, is showing efficacy in preclinical trials. Cases have also been reported to enter the clinical stage with regard to the treatment of some diseases. Exosomes released from stem cells are known to contain key factors associated with anti-inflammatory and self-renewal activities that stem cells retain.
Therefore, the therapy employing no cells has attracted intensive attention as a new approach capable of surmounting the disadvantages of existing cell therapies, such as securing and maintaining a therapeutically effective amount of cells.
However, as a rule, nucleated cells each release only about 1,000 exosomes. Therefore, it is very important in the therapeutic technology using exosomes to improve the yield of exosomes separated from cells.
On the whole, exosomes are obtained by separation from a cell culture. Stem cells are generally cultured in a 2D manner and the acquirement of a large amount of exosomes in such a manner needs culturing of a large amount of cells, resulting in an increase in cost.
In addition, the separation of exosomes from a large volume of the cell culture in which a large number of cells has been cultured requires considerable labor. Centrifugation and tangential flow filtration (TFF) are mainly used for separation and purification of exosomes.
Encountering the problem of limited applicable capacity, centrifugation is not suitable for separating exosomes from large volumes of cell cultures. TFF has the advantage of being suitable for a large-scale process compared to centrifugation, but suffers from various disadvantages including the shear stress generated during the filtration process, and loss of exosomes.
In order to solve this problem, there is a need for development of an efficient extraction method capable of obtaining a large amount of exosomes from a small number of cells and a small volume of a cell culture medium.
The present inventors have conducted thorough and intensive research to develop a method for efficiently obtaining mesenchymal stem cell-derived extracellular vesicles, especially extracellular vesicles derived from mesenchymal stem cells from the umbilical cord, which contain various beneficial components and are composed of a lipid bilayer, thus functioning as a stable drug delivery system.
As a result, mesenchymal stem cells derived from the umbilical cord (Wharton's jelly-derived MSCs, WJ-MSCs) were cultured in a medium containing Noxa protein-derived peptides in an orbital shaking culture manner to obtain floating single cells. The extracellular vesicles can be separated from the cell suspension at high yield with high purity and were found to exhibit stem cell-intrinsic wound healing and immunomodulatory effects.
Accordingly, an aspect of the present disclosure is to provide a method for producing mesenchymal stem cell-derived extracellular vesicles.
Another aspect of the present disclosure is to provide extracellular vesicles isolated from mesenchymal stem cells pretreated with a medium composition including a peptide composed of the amino acid sequence of SEQ ID NO: 1, glucose, sucrose, and MOPS, or a culture thereof.
A further aspect of the present disclosure is to provide a pharmaceutical composition including extracellular vesicles isolated from mesenchymal stem cells or a culture thereof for alleviation, suppression, or treatment of wound.
A yet further aspect of the present disclosure is to provide a pharmaceutical composition including extracellular vesicles isolated from mesenchymal stem cells or a culture thereof for alleviation, suppression, or treatment of inflammation.
A yet another aspect of the present disclosure is to provide a food composition including extracellular vesicles isolated from mesenchymal stem cells or a culture thereof for alleviation, suppression, or amelioration of an inflammatory disease.
The present disclosure is drawn to a method for mass production of extracellular vesicles, using a peptide derived from Noxa protein and mesenchymal stem cells derived from the umbilical cord and, specifically, to a method for mass production of extracellular vesicles, wherein mesenchymal stem cells are cultured in a medium composition containing a Noxa protein-derived peptide, glucose, sucrose, and 3-(N-morpholino)propanesulfonic acid (MOPS), whereby extracellular vesicles having wound healing and immunomodulatory effects can be obtained with high purity at high yield.
Below, a detailed description will be given of the present disclosure.
An aspect of the present disclosure pertains to a method for production of mesenchymal stem cell-derived extracellular vesicles, the method including:
In the present disclosure, the mesenchymal stem cells may be derived from at least one selected from the group consisting of bone marrow, embryo, umbilical cord, muscle, fat, and nerve tissue, for example, from the umbilical cord, but with no limitations thereto.
As used herein, the term “stem cells” refers to undifferentiated cells that self-renew and have the ability to differentiate into two or more different cell types.
In an embodiment of the present disclosure, the mesenchymal stem cells may be mesenchymal stem cells derived from the umbilical cord (Wharton's jelly-derived MSCs, WJ-MSCs).
The stem cells may be autologous or homologous stem cells, may be derived from any type of animals including human and non-human mammals, and may originate from an adult or an embryo, but with no limitations thereto.
In the present disclosure, the extracellular vesicles may be classified into the following three types according to the size and generation process thereof: Exosome; Apoptotic body; and Microveslcle (Ectosome).
As used herein, the term “exosome” refers to a cell-derived vesicle and exosomes are present in almost all eukaryotic body fluids.
In the present disclosure, exosomes each range in diameter from 30 to 100 nm, which is larger LDL proteins and much smaller than erythrocytes, but with no limitations thereto.
In the present disclosure, the peptide composed of the amino acid sequence of SEQ ID NO: 1 may be a Noxa protein-derived peptide.
The Bcl-2 homology 3 (BH3) domain of Noxa protein binds Mcl-1 and Bcl2A1 to inactivate their anti-apoptotic activities, which, in turn, activates BAX and BAK proteins, causing cytochrome-c to leak into the cytosol, where the caspase system completes the apoptotic process.
As used herein, the term “peptide” refers to a linear molecule which is formed as amino acid residues are bonded to each other via a peptide bond.
In one embodiment of the present disclosure, the Noxa protein-derived peptide may be a peptide having a homology of about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 98% or more, or about 99% or more with the peptide composed of the amino acid sequence of SEQ ID NO: 1.
The peptide in the present disclosure may be directly synthesized in a chemical manner using solid-phase peptide synthesis, for example, using an automated peptide synthesizer or may be biologically prepared by inserting DNA encoding the peptide a vector and expressing, but with no limitations thereto.
As used herein, the term “vector” refers to a means for expressing a gene of interest in a host cell.
Examples of the vector include plasmid vectors, cosmid vectors, and viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors, and adeno-associated virus vectors, but are not limited thereto.
The term “pre-culturing”, as used herein, refers to culturing mesenchymal stem cells to a predetermined level of confluency, for example, 50% or higher, 60% or higher, 70% or higher, 80% or higher, or 90% or higher confluency, but with no limitations thereto.
In an embodiment of the present disclosure, the first culturing step may further include a trypsin treatment step of treating the pre-cultured mesenchymal stem cells with trypsin to float the mesenchymal stem cells, but with no limitations thereto.
In an embodiment of the present disclosure, the first culturing step may further include an acquiring step of performing centrifugation to collect the pre-cultured mesenchymal stem cells, but with no limitations thereto.
In the present disclosure, the medium composition contains the peptide composed of the amino acid sequence of SEQ ID NO: 1 at a concentration of 0.1 to 5.0 uM, 0.1 to 4.5 uM, 0.1 to 4.0 uM, 0.1 to 3.5 uM, 0.1 to 3.0 uM, 0.1 to 2.5 uM, 0.1 to 2.0 uM, 0.1 to 1.5 uM, 0.5 to 5.0 uM, 0.5 to 4.5 uM, 0.5 to 4.0 uM, 0.5 to 3.5 uM, 0.5 to 3.0 uM, 0.5 to 2.5 uM, 0.5 to 2.0 uM, or 0.5 to 1.5 uM, and for example, 0.5 to 1.5 uM, but with no limitations thereto.
In the present disclosure, the medium composition contains glucose at a concentration of 1 to 10 mM, 1 to 8 mM, 1 to 6 mM, 2 to 10 mM, 2 to 8 mM, 2 to 6 mM, 3 to 10 mM, 3 to 8 mM, 3 to 6 mM, 4 to 10 mM, 4 to 8 mM, or 4 to 6 mM, for example, 4 to 6 mM, but with no limitations thereto.
In the present disclosure, the medium composition contains sucrose at a concentration of 200 to 300 mM, 200 to 280 mM, 200 to 260 mM, 220 to 300 mM, 220 to 280 mM, 220 to 260 mM, 240 to 300 mM, 240 to 280 mM, or 240 to 260 mM, for example, 240 to 260 mM, but with no limitations thereto.
In the present disclosure, the medium composition contains 3-(N-morpholino)propanesulfonic acid [MOPS] at a concentration of 1 to 20 mM, 1 to 18 mM, 1 to 16 mM, 1 to 14 mM, 1 to 12 mM, 3 to 20 mM, 3 to 18 mM, 3 to 16 mM, 3 to 14 mM, 3 to 12 mM, 5 to 20 mM, 5 to 18 mM, 5 to 16 mM, 5 to 14 mM, 5 to 12 mM, 8 to 20 mM, 8 to 18 mM, 8 to 16 mM, 8 to 14 mM, or 8 to 12 mM, for example, 8 to 12 mM, but with no limitations thereto.
In the present disclosure, the second culturing step is adapted for culturing the pre-cultured mesenchymal stem cells in a medium composition including a peptide consisting of the amino acid sequence of SEQ ID NO: 1, glucose, sucrose, and 3-(N-morpholino)propanesulfonic acid [MOPS], but with no limitations thereto.
In the present disclosure, the second culturing step may be carried out for 5 to 30 minutes, 5 to 25 minutes, 5 to 20 minutes, 5 to 18 minutes, 10 to 30 minutes, 10 to 25 minutes, 10 to 20 minutes, 10 to 18 minutes, 13 to 30 minutes, 13 to 25 minutes, 13 to 20 minutes, or 13 to 18 minutes, for example, 13 to 18 minutes, but with no limitations thereto.
In an embodiment of the present disclosure, the second culturing step may be performed in an orbital shaking culture manner.
As used herein, the term “orbital shaking culture” refers to culturing cells in, for example, a flask on a plate that is horizontally rotating in a circle of a predetermined radius at a constant speed, but with no limitations thereto.
In an embodiment of the present disclosure, the orbital shaking culture may be performed using an orbital shaker.
In an embodiment of the present disclosure, the production method may further include an isolation step of isolating the mesenchymal stem cell-derived extracellular vesicles, but with no limitations thereto.
In the present disclosure, the isolation step may be adapted for obtaining extracellular vesicles from the medium composition that has undergone the second culturing step, but with no limitations thereto.
In an example of the present disclosure, when cultured in the medium composition containing a peptide composed of the amino acid sequence of SEQ ID NO: 1, glucose, sucrose, and MOPS, mesenchymal stem cells were measured to produce an increased number of total particles, compared to the control (Table 8).
In an embodiment of the present disclosure, when cultured in an orbital shaking culture manner in the medium composition containing a peptide composed of the amino acid sequence of SEQ ID NO: 1, glucose, sucrose, and MOPS, mesenchymal stem cells were measured to produce an increased number of total particles, compared to the control or those cultured in a non-orbital shaking culture manner (Table 8).
In an embodiment of the present disclosure, when cultured in an orbital shaking culture manner in the medium composition containing a peptide composed of the amino acid sequence of SEQ ID NO: 1, glucose, sucrose, and MOPS, mesenchymal stem cells were measured to produce an increased number of total particles, compared to the control or those cultured in an orbital shaking culture manner or a non-orbital shaking culture manner (Table 8).
The method for producing extracellular vesicles according to the present disclosure can produce extracellular vesicles at high yield with high purity. In an embodiment of the present disclosure, the extracellular vesicles produced when culturing mesenchymal stem cells in an orbital shaking culture manner in the medium composition containing a peptide composed of the amino acid sequence of SEQ ID NO: 1, glucose, sucrose, and MOPS were observed to have greatly increased purity, compared to the control (
Another aspect of the present disclosure is drawn to extracellular vesicles derived from mesenchymal stem cells pretreated with a peptide composed of the amino acid sequence of SEQ ID NO: 1, glucose, sucrose, and 3-(N-morpholino)propanesulfonic acid [MOPS].
In the present disclosure, the pre-treatment may refer to a process of treating stem cells with trypsin in advance.
In an embodiment of the present disclosure, the mesenchymal stem cells may be further pretreated with a medium composition containing a peptide composed of the amino acid sequence of SEQ ID NO: 1, glucose, sucrose, and MOPS.
In the present disclosure, the term “culture” may mean a culture supernatant after mesenchymal stem cells are cultured in a typical manner known in the art or a cell-suspended culture medium after mesenchymal stem cell are cultured in an orbital shaking culture manner, but with no limitations thereto.
The extracellular vesicles of the present disclosure may exhibit a palliative, suppressive, or healing effect on wound.
In an embodiment of the present disclosure, the extracellular vesicles isolated from mesenchymal stem cells pretreated with the medium composition or from a culture thereof were observed to exhibit relatively improved cell migration, compared to the control (
Extracellular vesicles isolated from the mesenchymal stem cells or a culture thereof can exhibit a palliative, suppressive, or therapeutic effect on inflammatory diseases.
In an embodiment of the present disclosure, extracellular vesicles isolated from mesenchymal stem cells pretreated with the medium composition or a culture thereof are observed to reduce the level of nitric oxide (NO) and downregulate the mRNA expression of inflammation-inducing genes (iNOS, TNF-α, IL-1β, IL-6, COX-2) in LPS-induced inflammation models (
Another aspect of the present disclosure is concerned with a pharmaceutical composition including extracellular vesicles derived from mesenchymal stem cells pretreated with a peptide composed of the amino acid sequence of SEQ ID NO: 1, glucose, sucrose, and 3-(N-morpholino)propanesulfonic acid [MOPS] for palliating, suppressing, or healing a wound.
As used herein, the term “wound” refers to a damaged site present in damaged tissues such as skin, organs, or bones.
In the present disclosure, the palliation, suppression, or healing of a wound may mean mitigating or inhibiting the degree of tissue damage or therapeutically treating a damaged tissue by, for example, a method of promoting cell differentiation in a damaged tissue, but with no limitations thereto.
In the present disclosure, the pharmaceutical composition for palliating, suppressing, or healing a wound may be a cell therapy product.
As used herein, the term “cell therapy product” refers to a therapeutic, diagnostic, and prophylactic medicine designed to restore the organization of cells and tissues through a series of actions responsible for modifying biological traits of cells by proliferating and selecting living autologous, allogenic, or xenogenic cells or by other methods.
In one embodiment of the present disclosure, the cell therapy product may be a stem cell therapy product.
In the present disclosure, the term “stem cell therapy product” may refer to a biopharmaceutical using autologous bone marrow-derived, autologous adipocyte-derived, or allogeneic cord blood-derived stem cells.
In the present disclosure, the pharmaceutical composition may include, as an active ingredient, exosomes isolated from mesenchymal stem cells or a culture thereof.
In the present disclosure, the pharmaceutical composition may include, as an active ingredient, an extracellular vesicle isolated from a mesenchymal stem cell pretreated with a peptide composed or the amino acid sequence of SEQ ID NO: 1, glucose, sucrose, and MOPS or from a culture thereof.
As used herein, the term “including as an active ingredient” means including extracellular vesicles isolated from stem cells or a culture thereof in an amount sufficient to achieve alleviation, inhibition, or therapeutic activity for a specific disease.
In the present disclosure, the pharmaceutical composition may include a pharmaceutically acceptable carrier, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, etc., but is not limited thereto.
In the present disclosure, the pharmaceutical composition may further include a lubricant, a humectant, a sweetener agent, a flavorant, an emulsifier, a suspending agent, and a preservative, but is not limited thereto.
In the present disclosure, the pharmaceutical composition can be administered orally and parenterally, for example, through intravenous, subcutaneous, intramuscular, intraperitoneal, topical, intranasal, intrapulmonary, rectal, intrathecal, intraocular, dermal, and transdermal routes, but with no limitations thereto.
In the present disclosure, the administration dose of the pharmaceutical composition is determined to effectively achieve desired palliation, suppression, and therapy and may vary depending on various factors including formulation methods, administration modes, patient's age, weight, sex, pathological condition, and meal, the time of administration, the route of administration, excretion rates, and response sensitivity. For example, the daily dosage of the pharmaceutical composition of the present disclosure may be 0.0001-1000 mg/kg.
In the present disclosure, according to any conventional method easily implementable by one of ordinary skill in the art, the pharmaceutical composition may be formulated into a unit dosage form or enclosed in a multi-dose container, together with a pharmaceutically acceptable carrier and/or excipient. In this regard, the formulation may be a solution, a suspension, or an emulsion in oil or aqueous media, or a syrup, an extract, a pulvis, a granule, a tablet, or a capsule, which may further include a dispersing agent or a stabilizer, but with no limitations thereto.
Another aspect of the present disclosure is drawn to a pharmaceutical composition including extracellular vesicles derived from mesenchymal stem cells pretreated with a peptide composed of the amino acid sequence of SEQ ID NO: 1, glucose, sucrose, and 3-(N-morpholino)propanesulfonic acid [MOPS] for alleviation, suppression, or treatment of an inflammatory disease.
In the present disclosure, the inflammatory disease may be at least one selected from the group consisting of atopic dermatitis, edema, dermatitis, allergy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, parotid arthritis, tendinitis, tendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, and multiple sclerosis, but with no limitations thereto.
Another aspect of the present disclosure is drawn to a food composition including extracellular vesicles derived from mesenchymal stem cells pretreated with a peptide composed of the amino acid sequence of SEQ ID NO: 1, glucose, sucrose, and 3-(N-morpholino)propanesulfonic acid [MOPS].
In the present disclosure, the food composition may be a food composition for alleviation, suppression, or palliation of an inflammatory disease.
In the present disclosure, the food composition may include ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents, but with no limitations thereto.
In the present disclosure, examples of the carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose, sucrose, and oligosaccharides, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol, but are not limited thereto.
Examples of the flavoring agent according to the present disclosure include, but are not limited to, natural flavoring agents such as thaumatin and stevia extract, and synthetic flavoring agents such as saccharin and aspartame.
According to the production method of the present disclosure, extracellular vesicles having wound healing and immunomodulatory effects can be obtained at high yield with high purity by culturing mesenchymal stem cells in an orbital shaking mode in a medium composition containing a noxa protein-derived peptide (peptide), glucose, sucrose, and 3-(N-morpholino)propanesulfonic acid [MOPS].
In addition, the extracellular vesicles derived from mesenchymal stem cells pretreated with a medium composition containing a noxa protein-derived peptide, glucose, sucrose and MOPS of the present disclosure exhibit excellent wound palliation, suppression, or healing effect.
Furthermore, the extracellular vesicles derived from mesenchymal stem cells pretreated with a medium composition containing a noxa protein-derived peptide, glucose, sucrose and MOPS of the present disclosure exhibit an excellent alleviative, suppressive, or therapeutic effect on inflammatory diseases.
A method for production of mesenchymal stem cell-derived extracellular vesicles, the method including:
a first culturing step of pre-culturing mesenchymal stem cells; and
a second culturing step of culturing the pre-cultured mesenchymal stem cells in a medium composition containing a peptide composed of the amino acid sequence of SEQ ID NO: 1, glucose, sucrose, and 3-(N-morpholino)propanesulfonic acid [MOPS].
A better understanding of the present disclosure may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present disclosure.
Mesenchymal stem cells derived from the umbilical cord (Wharton's jelly-derived MSCs, WJ-MSCs) were assayed for viability by treatment with the eMTD peptide at predetermined concentrations for predetermined periods of time.
1-1. Absorbance Analysis
Tests were conducted with predetermined concentrations of the eMTD peptide. In this regard, umbilical cord-derived mesenchymal stem cells were seeded at a density of 2×104 cells/well into 24-well plates (30024, SPL). After 24 hours of incubation, the eMTD peptide in a sucrose buffer was applied at a final concentration of 0, 0.5, 1, 3, 5, 10, or 20 μM to the cells.
The sequence of the eMTD peptide is given in Table 1.
The sucrose buffer contained glucose, sucrose, and 3-(N-morpholino)propanesulfonic acid [MOPS] at the respective concentrations shown in Table 2.
After 15 minutes, the cells were incubated with ez-cytox (EZ-3000, DOGEN) for 30 to 60 minutes. Then, absorbance was read at 450 nm using a Bio-RAD x-Mark™ spectrophotometer (Bio-Rad Laboratories, USA).
In addition, treatment with the eMTD peptide was conducted over various times. In this regard, umbilical cord-derived mesenchymal stem cells were seeded at a density of 2×10 4 cells/well into 24-well plates and incubated for 24 hours. Thereafter, the cells were treated with 1 μM of the eMTD peptide in the sucrose buffer for various periods of time (0, 5, 10, 15, 20, 25, or 30 minutes).
After 15 minutes of treatment, the cells were incubated for 30 to 60 minutes with ez-cytox (EZ-3000, DOGEN). Absorbance was read at 450 nm using the Bio-RAD x-Mark™ spectrophotometer (Bio-Rad Laboratories, USA). The results are depicted in
1-2. Cell Counting
To measure the number of cells, umbilical cord-derived mesenchymal stem cells were seeded at a density of 2.5×104 cells/well into 12-well plates (30012, SPL) and incubated for 24 hours. The eMTD peptide in the sucrose buffer of Table 2 was applied at a final concentration of 0, 0.5, 1.0, 3.0, 5.0, 10.0, or 20.0 μM to the cells. After 15 minutes, the cells were counted using a trypan blue solution.
Separately, umbilical cord-derived mesenchymal stem cells were seeded at a density of 2.5×104 cells/well into 12-well plates (30012, SPL) and incubated for 24 hours. The cells were treated with the eMTD peptide in the sucrose buffer of Table 2 for various periods of time (0, 5, 10, 15, 20, 25, and 30 minutes). After each period, the cells were counted using a trypan blue solution, and the results are depicted in
Referring to data in
2-1. Preparation of Control-EV
Umbilical cord-derived mesenchymal stem cells were seeded into 150-mm dishes (20151, SPL) (5000 cells/cm 2). When the cells were grown to 80 to 90% confluence, the medium was changed with a-MEM (a-Minimum Essential Medium) (12561072, Gibco) supplemented with 10% exosome-depleted FBS (PS-FB1, PEAK).
After 48 hours of incubation, the a-MEM medium of the culture was obtained. Cell debris was removed by briefly spinning at 300 g for 3 minutes, followed by centrifugation at 2,000 g for 10 minutes. The supernatant was transferred to a new tube which was then again centrifuged at 10,000 g for 30 minutes. The resulting supernatant was again subjected to centrifugation at 187,000 g for 2 hours to obtain Control-EV as a pellet.
2-2. Preparation of eEV (eMTD-EV)
The pellet obtained according to Experimental Example 2-1 was incubated for 15 minutes with the eMTD peptide (1 μM) in the sucrose buffer, followed by isolating eEV (eMTD-EV).
2-3. Preparation of S-eEV (Shaking-eMTD-EV)
The pellet obtained according to Experimental Example 2-1 was incubated for 15 minutes with the eMTD peptide (1 μM) in the sucrose buffer using an orbital shaker (60 RPM) (69455, INFORS HT Celltron), followed by isolating S-eEV (shaking-eMTD-EV).
2-4. Preparation of T-eEV (Trypsinization-eMTD-EV)
When reaching 80 to 90% confluence according to the procedure of Experimental Example 2-1, the cells were floated using trypsin (25200-056, Gibco). Centrifugation of the cell suspension gave a cell pellet which was then incubated for 15 minutes with the eMTD peptide (1 μM) in the sucrose buffer in a 50-ml conical tube (50050, SPL), followed by isolating T-eEV (Trypsinization-eMTD-EV).
2-5. Preparation of TS-eEV (Trypsinization Shaking-eMTD-EV)
The cells were floated according to the procedure of Experimental Example 2-4 and then centrifuged. The cell pellet thus obtained was treated with the eMTD peptide (1 μM) in the sucrose buffer in a 50-ml conical tube (50050, SPL).
The treatment was continued for 15 minutes using an orbital shaker (60 RPM) before isolation of TS-eEV (Trypsinization shaking-eMTD-EV).
2-5. EV Isolation and Comparison of Production Output
The umbilical cord-derived mesenchymal stem cells from which Control-EV, eEV, S-eEV, T-eEV, and TS-eEV have been isolated are summarized for starting cell counts and harvested cell counts, together with incubation times needed to obtain extracellular vesicles, in Table 7.
The resulting Control-EV, eEV, S-eEV, T-eEV, and TS-eEV were counted as shown in
5.45 × 10−1 ± 2.99 × 10−1
As can be seen in
S-eEV was counted 4.89×1010±2.14×109, with about +310.9% improvement compared to Control-EV, T-eEV and TS-eEV were counted 7.30×1010±1.74×109 and 2.64×1011±3.78×109, which were improved by about +513.4% and about +2,118.5%, compared to Control-EV, respectively.
Specially, costs for acquiring 1×1010 EVs are given in Table 9. TS-eEV was calculated to be about 70 times cheaper than Control-EV.
, won)
1-1. Analysis for EV Size and Particle Number by EV Size
The size of EVs was measured by dynamic light scattering (DLS) analysis using a Nano Zetasizer (Malvern Instruments, Melbourne, UK). EVs were measured for size and counted using a nanoparticle tracking analyzer (NTA) NS300 (Nanosight, Amesbery, UK). The results are depicted in
As shown in
1-2. Morphological Analysis for EV
EVs were morphologically analyzed using a transmission electron microscope (TEM, JEM-1010, Nippon Denshi, Tokyo, Japan) at 80 kV, and the results are given in
As shown in
1-3. Analysis for EV Purity
Particle numbers were determined using NTA. Further, proteins were quantitated using a BCA kit, followed by measuring purity of EV (particles/ug protein). The results are given in
3.22 × 108 ± 1.22 × 108
As shown in
Control-EV was obtained by culturing cells and thus was highly likely to be in mixture with various substances secreted from cells, such as soluble proteins or cytokines.
1-4. Identification of Surface Marker on EV
The EVs were captured using Exosome-Human CD9 Flow Detection Reagent (Invitrogen, 10620D), and dyed with CD9-BV421 (743047, BD), CD63-PE (556020, BD), or CD81-APC (130-119-787, Miltenyi Biotec), followed by flow cytometry (Beckman Coulter/CytoFLEX). The results are depicted in
As shown in
Umbilical cord-mesenchymal stem cells were lysed with a RIPA buffer (CBR002, LPS solution) containing a protease inhibitor cocktail (87786, Invitrogen) to give a whole cell lysate (WCL). WCL and EV were run in 4-12% Bis-Tris Flus Gels (NW04125BOX, Invitrogen/NW04122BOX, Invitrogen) by electrophoresis and then transferred onto an NC membrane (IB23001, Invitrogen). The NC membrane was incubated overnight at 4° C. with a primary antibody (1:1000) and washed three times with 1×TBST (TLP-118.1, Translab). Subsequently, the membrane was reacted at room temperature for 2 hours with a secondary antibody before being washing with 1×TBST.
The primary and secondary antibodies were as follows: anti-CD9 antibody (ab263023, Abcam), anti-CD63 antibody (ab134045, Abcam), anti-HSP70 antibody (4876, CST), anti-Flotillin-1 antibody (18634, CST), anti-Alix antibody (2171, CST), anti-GM130 antibody (12480, CST), β-actin antibody (sc-47778, Santa Cruz), HRP linked anti-rabbit IgG (7074, CST), and HRP linked anti-mouse IgG (7076, CST).
All the antibodies were diluted in 1× blocking buffer (TLP-115.1G, Translab) before use, and images were taken using Invitrogen™ iBright™ Imagers (CL-1000). The results are depicted in
As can be seen in
1-5. EV Uptake of HaCaT Cells
EVs were dyed with 2 μg/ml DiR (D12731, Invitrogen) at room temperature for one hour. Thereafter, free dye was removed using an ultracentrifuge at 178,000×g for two hours. HaCaT cells were incubated with 3×109 EVs for six hours and then stained with DAPI (4′, 6-diamidino-2-phenylindole) [VECTASHIELD® Antifade Mounting Medium with DAPI-(H-1200)] and CellMask™ (Green Plasma Membrane Stain, C37608, Invitrogen). The stained HaCaT cells were observed by confocal laser microscopy (Carl Zeiss LSM 800), and the results are depicted in
As shown in
2-1. Viability Test
HaCaT cells were tested for viability against Control-EV and TS-eEV. In this regard, HaCaT cells were seeded at a density of 1×104 cells/well into 96-well plates (30096, SPL). After 24 hours of incubation, the medium was changed with DMEM-high glucose (D6046, Sigma) supplemented with 10% exosome-depleted FBS and treated for 24 hours with predetermined numbers of EVs (1×106, 1×107, 1×108, or 1×109 particles). After 24 hours, the cells were incubated for one hour with ez-cytox (EZ-3000, DOGEN). Absorbance was read at 450 nm using Bio-RAD x-Mark™ spectrophotometer (Bio-Rad Laboratories, USA). The results are depicted in
As understood from the data of
2-2. Cell Migration and Proliferation Effect
In order to examine the effects of Control-EV and TS-eEV on cell migration and proliferation, HaCaT cells were seeded at a density of 6×105 cells/well into 6-well plates (32006, SPL) and grown to 95% confluency, followed by incubation with 10 μg/ml mitomycin C (M4287, Sigma) for two hours to inhibit cell proliferation.
The cells were scratched using a 1-ml pipette tip and treated with Control-EV or TS-eEV (1×109 particles/ml) for 24, 48, or 72 hours. The effects of Control-EV and TS-eEV on cell migration and proliferation were observed under a microscope. The resulting images are given in
In Table 12, relative wound areas for Con-EV and TS-eEV over time are numerically expressed, with normalization to the value 100 set for the control of
As can be seen in
There was no significant difference between Control-EV and TS-eEV, and treatment with 1×109 particles of TS-eEV increased cell migration by about +27.0% or more, compared to the control (PBS).
2-3. Anti-Inflammatory Effect
Raw264.7 cells were seeded at a density of 1.5×105 cells into 24-well plates and incubated for 12 hours. Then, 1×108 or 1×109 particles of Control-EV was applied, together with 10 ng/ml LPS (L4391-1MG, Sigma), to 300 μl of the Raw264.7 cell culture. In addition, 1×108 or 1×109 particles of TS-eEV, instead of Control-EV, were applied to 300 μl of the Raw264.7 cell culture.
After 18 hours of incubation, the culture supernatant was obtained and reacted with the Griess reagent [0.1% N-(1-naphthyl) ethylenediamide dihydrochloride and 1% sulfanilamide in 5% phosphoric acid], followed by reading absorbance at 540 nm to quantitatively analyze nitric oxide (NO). The results are depicted in
As can be seen in
The NO level was measured to be 15.0±0.20 μM upon treatment with 1×109 particles of TS-eEV and thus decreased by about −45.8%, compared to 21.7±0.4 μM measured for LPS only.
The expression level of iNOS, an inflammation mediator, was measured to be 1.46±0.08 and thus decreased by about −45.7%, compared to 2.69±0.27 measured for LPS only.
The expression level of TNF-α was measured to be 1.46±0.07 and thus decreased by about −36.0%, compared to 2.28±0.57 measured for LPS only.
The expression level of IL-113 was measured to be 1.51±0.13 and thus decreased by about −63.9%, compared to 4.18±0.52 measured for LPS only.
The expression level of IL-6 was measured to be 0.71±0.04 and thus decreased by about −56.2%, compared to 1.62±0.1 measured for LPS only.
The expression level of COX2 was measured to be 1.56±0.22 and thus decreased by about −45.6%, compared to 2.87measured for LPS only.
As understood from the data, the LPS (Lipopolysaccharide)-induced inflammatory response in Raw 264.7 (mouse macrophage) cells can be reduced by TS-eEV, which demonstrates the anti-inflammatory effect of TS-eEV.
BALB/c nude female mice at 6 weeks of age were purchased and acclimated for one week. After a tissue wound was induced using a 5-mm biopsy punch (Kai, BP-50F), Control-EV or TS-eEV was applied at a concentration of 1×109 particles/20 μl to the wound site that was monitored every day by photography to determine wound areas. The results are depicted in
As can be seen in
The cells were pretreated with 50 μM BAPTA-AM (calcium-selective chelator) for 1 hour and with 10 μM ALLM (calpain inhibitor) for 18 hours. Comparison was made of EV production outputs among the method used, and the results are shown in
As understood from the data in
The present disclosure relates to a method for mass production of extracellular vesicles, using a peptide derived from Noxa protein and mesenchymal stem cells and, specifically, a method for mass production of extracellular vesicles, wherein mesenchymal stem cells are cultured in a medium composition containing a Noxa protein-derived peptide, glucose, sucrose, and 3-(N-morpholino)propanesulfonic acid (MOPS), whereby extracellular vesicles having wound healing and immunomodulatory effects can be obtained with high purity at high yield.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0174891 | Dec 2020 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/018761 | 12/10/2021 | WO |
