WO2020251347A1

WO2020251347A1 - A method of preparing exosomes from mesenchymal stem cells

Info

Publication number: WO2020251347A1
Application number: PCT/MY2020/050039
Authority: WO
Inventors: See Nguan NG; Mimi Gim Ewe LEE
Original assignee: Ming Medical Sdn Bhd
Priority date: 2019-06-13
Filing date: 2020-06-12
Publication date: 2020-12-17

Abstract

The present invention relates to a method of preparing exosomes having a particle size of 50-250 nm from mesenchymal stem cells, the method comprising the step of providing a cell culture supernatant from the mesenchymal stem cells-conditioned cell culture medium, the cell culture supernatant comprising exosomes; contacting the supernatant with polyethylene glycol having a molecular weight of 2,000-6,000 daltons to isolate and purify the exosomes; and further purifying the isolated exosomes via the method of polyethylene glycol precipitation, wherein the polyethylene glycol has a molecular weight of 4,000-12,000 daltons.

Description

A METHOD OF PREPARING EXOSOMES FROM MESENCHYMAL STEM

CELLS

FIELD OF INVENTION

The present invention relates to a method for preparing exosomes from mesenchymal stem cells (MSCs). Particularly, the preparation method comprises several cycles of polyethylene glycol (PEG) precipitation for purification of the exosomes. More particularly, the PEG of different molecular weights may be employed in each cycle to substantially remove impurities.

BACKGROUND OF THE INVENTION

Exosomes are cell-derived vesicles. They are found in biological fluids, such as urine, plasma and ascites. The exosomes are generated by inward budding of endosomal multivesicular bodies. The cargo of exosomes includes proteins/glycoproteins expressed on the cell membrane as well as molecules and soluble factors present in the cytosol of parental cells.

The exosomes represent a next generation therapeutic platform for regenerative medicine. These nano-sized extracellular membranous vesicles are potent delivery vehicles for functional messenger RNA (mRNA), miRNA and DNA molecules as well as proteins. There is growing evidence suggesting that they can impart similar therapeutic benefits as the producer cells.

The exosomes produced from MSCs is able to exert modulating effects on tissue repair and regeneration in heart, kidney, skin and liver. The MSCs-derived exosomes promote hepatic regeneration in drug-induced liver injury models. Moreover, the exosomes released from human induced pluripotent stem cells-derived MSCs is able to facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. It has also been demonstrated that exosomes containing particular miRNAs is able to limit fibrosis, modulate immune response, stimulate cell proliferation, spur angiogenesis, and improve functional recovery.

The nano-sized exosomes have major manufacturing and toxicology advantages over cells such as the ability to increase sterility assurance in the process of using microbial retentive filters (e.g. 0.22 pm). The exosomes as non-living carriers present potentially lower risk for adverse tumorigenic and immunogenic response. Besides, the exosomes also possess more flexibility in terms of stable drug storage as compared to cells. For example, the exosomes can be conveniently stored at room and cold temperatures whereas the cells require storage in liquid nitrogen. Therefore, there exists a need to efficiently produce high yield of exosomes for commercial purposes. In general, the method of generating MSCs derived-exosomes firstly involves the step of seeding and growing the MSCs to confluence in fetal bovine serum (FBS)- containing cell culture medium. For exosome production, the confluent layer of MSCs are then washed and cultured under serum-free and normoxic (20% oxygen) conditions for 48 hours. The exosomes are later isolated from the conditioned medium using a precipitation method, followed by formulating the isolated exosomes in a base serum-free medium. However, the exosomes produced from such method lacks the desirable purity and stability for pharmaceutical or therapeutic use.

Thus, there is a need to provide an improved preparation method for producing exosomes from MSCs that is able to overcome the drawback aforementioned.

SUMMARY OF INVENTION

The main aspect of the present invention is to provide a method that enables preparation of highly purified and stable exosome from MSCs. Preferably, the exosome prepared is suitable for use in pharmaceutical and therapeutic applications.

At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention is a method of preparing exosomes having a particle size of 50-250 nm from MSCs, the method comprising the step of providing a cell culture supernatant from the MSCs- conditioned cell culture medium, the cell culture supernatant comprising exosomes; contacting the supernatant with PEG having a molecular weight of 2,000-6,000 daltons to isolate and purify the exosomes; and further purifying the isolated exosomes via the method of PEG precipitation, wherein the PEG has a molecular weight of 4,000-12,000 daltons.

In accordance with a preferred embodiment, the MSCs are derived from cardiac tissue, adipose tissue, neural tissue, insulin producing cells or umbilical cord (UC) tissue.

In another preferred embodiment, the MSCs are cultured in the cell culture medium at a dissolved carbon dioxide (CO2) concentration of between 1-19%.

Preferably, the cell culture medium is selected from knockout DMEM (KO-DMEM) or MesenCultTM-hPL.

It is preferable that the exosomes prepared from the method aforementioned has a protein content of 0.5-100 pg.

Preferably, the exosomes prepared from the method aforementioned is positive for at least one of the cellular exosome markers consisting of the group of CD9, CD63, CD81, TSG101, Annexins and HSP70. Advantageously, the exosomes prepared from the method aforementioned is negative for at least one of the cellular exosome markers consisting of the group of GRP94 and Calnexin.

The present invention also provides a pharmaceutical composition comprising the exosomes aforementioned.

The present preferred embodiment of the invention consists of novel features and a combination of parts hereinafter fully described and illustrated in the accompanying drawings and particularly pointed out in the appended claims; it being understood that various changes in the details may be effected by those skilled in the arts but without departing from the scope of the invention or sacrificing any of the advantages of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction and operation and many of its advantages would be readily understood and appreciated.

Figures lA and IB depict the characterization of MSCs from UC. Morphology of UC- MSCs, source of the exosomes showed fibroblast-like morphology. Scale bar, 100 pm.

Figure 2 depicts the characterization of MSCs derived from UC using flow cytometry. Flow cytometric analysis of cell surface markers of UC-MSCs expressed positive markers of CD73, CD90, CD105 and CD44 but not CD34, CDllb, CD19, CD45 and HLA-DR which are negative markers.

Figure 3 depicts the differentiation potential of MSCs isolated from UC. (A) Adipogenesis of UC-MSCs. The cells were stained with Oil Red O after 3 weeks in an induction culture. The lipid globules of the adipocytes were stained red. Scale bar, 100 pm. (B) Osteogenesis of UC-MSCs. Cells were stained with Von Kossa after osteogenic induction for 3 weeks. The calcium deposits of the osteocytes were stained in brown. Scale bar, 100 pm. (C) Chondrogenesis of UC-MSCs. The cells were stained with Alcian Blue after chondrogenic induction for 3 weeks. The proteoglycans deposits of chrondrocytes were stained blue. Scale bar, 25 pm.

Figure 4 depicts the size distributions of (A) pre-lyophilized (widely distributed from 30 to 400 nm and peaks at 102 nm) and (B) post-lyophilized (widely distributed from 50 to 600 nm and peaks at 184 nm) UC-MSCs-derived exosomes using Dynamic Light Scattering (DLS) system.

Figure 5 depicts the Zeta potential (z-potential) of (A) pre-lyophilized (-13.7 mV) and (B) post-lyophilized (-16.5 mV) MSCs-derived exosomes using Zetasizer analysis.

Figure 6 depicts the total protein expression of pre-lyophilized and post-lyophilized MSCs-derived exosomes using Western Blot.

Figure 7 depicts the marker expression of post-lyophilized MSCs-derived exosomes using Western Blot. (A) All MSCs-derived exosomes were enriched with tetraspanins positive markers CD63. (B) All MSCs-derived exosomes were enriched with endosomes positive markers TSG101. (C) All MSCs-derived exosomes showed the absence of endoplasmic reticulum negative markers GRP94.

DETAILED DESCRIPTION OF THE INVENTION Hereinafter, the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawings. However, it is to be understood that limiting the description to the preferred embodiments of the invention and to the drawings is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.

The present invention relates to a method of preparing exosomes having a particle size of 50-250 nm from MSCs, the method comprising the step of providing a cell culture supernatant from the MSCs-conditioned cell culture medium, the cell culture supernatant comprising exosomes; contacting the supernatant with PEG having a molecular weight of 2,000-6,000 daltons to isolate and purify the exosomes; and further purifying the isolated exosomes via the method of PEG precipitation, wherein the PEG has a molecular weight of 4,000-12,000 daltons.

In accordance with a preferred embodiment, the MSCs are derived from cardiac tissue, adipose tissue, neural tissue, insulin producing cells or UC tissue.

In the step of culturing cell, the MSCs are preferably grown to achieve an amount of about 10⁴-10^u cells. To achieve cell confluency, the MSCs are preferably cultured in the cell culture medium for about 2-20 days.

The MSCs used are preferably primary cells. The primary cells can be at least at passage number 2-10. Immortalized cells are also encompassed by the invention.

The MSCs are preferably cultured in a cell culture medium selected from KO-DMEM or MesenCult™-hPL. It is also preferable that the MSCs are cultured in the cell culture medium at a dissolved CO2 concentration of between 1-19%. More preferably, the MSCs are cultured under a dissolved CO2 concentration of between 2-7%. In a first exemplary embodiment, the MSCs can be cultured in KO-DMEM containing 5- 20% of FBS under about 5% CO2 at about 37 °C. In a second exemplary embodiment, the MSCs can be cultured in MesenCult™-hPL containing 1-5% or without antibiotic under about 5% CO2 at about 37 °C.

To isolate the exosome from the MSCs-conditioned medium, the cell culture supernatant comprising the exosome is subjected to the method of PEG precipitation. The cell culture supernatant comprising the exosome is preferably contacted with the PEG having a molecular weight of 2,000-6,000 daltons to isolate and purify the exosomes. More preferably, the PEG with molecular weight of about 4,000 daltons is employed for isolating and purifying the exosomes. In another embodiment, the PEG is used at a final concentration of 5-12% for precipitating and isolating the exosomes. Although PEG is preferred to be used in the present invention, other precipitating agent such as EXOQUICK® may also be considered.

While the first step of PEG precipitation may remove substantially large amount of cell debris from the exosomes, other impurities including but not limited to dead cells, apoptotic bodies and microbial contaminants may still remain therewith. To solve the problem aforementioned, a second step of PEG precipitation is performed to further purify the exosomes. In the preferred embodiment, different molecular weight of PEG is employed in the step of further purification. In accordance with the preferred embodiment, the PEG having a molecular weight of 4,000-12,000 daltons, is used to further remove the remaining impurities in the exosomes. According to a preferred embodiment, the exosomes may be subjected to third or fourth step of PEG precipitation for further purification. Preferably, PEG of different molecular weights may be employed in each of the following steps of further purification.

According to the preferred embodiment, the exosome prepared from the method aforementioned has a particle size of 50-250 nm. In one embodiment, the exosome prepared from the method aforementioned has a protein content of 0.5-100 pg. Preferably, the exosome prepared from the method aforementioned is positive for at least one of the cellular exosome markers consisting of the group of CD9, CD63, CD81, TSG101, Annexins and HSP70. Advantageously, the exosome prepared from the method aforementioned is negative for at least one of the cellular exosome markers consisting of the group of GRP94 and Calnexin.

The purified exosomes prepared from the method aforementioned is relatively stable to be stored at a low temperature under the condition of sublimation. Preferably, the storage temperature is maintained at between from -80 °C to -20 °C.

The purified exosomes may be provided in a solution of phosphate buffer saline or PLASMALYTE. In one embodiment, the purified exosomes are lyophilized at a temperature of about -70 °C prior to sublimation. In another embodiment, the purified exosomes are mixed with about 1-6 % of lyoprotectant selected from mannitol, lactose, sucrose or trehalose prior to lyophilization. In yet another embodiment, the lyophilized exosomes are subjected to a vacuum treatment at a temperature of about - 70 °C.

For further use, the lyophilized exosomes can be resuspended in an aqueous solution, preferably water or saline. The exosomes can be used for preparation of a lyophilized or a liquid formulation. The formulation comprising the exosomes aforementioned is suitable for use in pharmaceutical and therapeutic application.

Although the invention has been described and illustrated in detail, it is to be understood that the same is by the way of illustration and example, and is not to be taken by way of limitation. The scope of the present invention is to be limited only by the terms of the appended claims.

Claims

1. A method of preparing exosomes having a particle size of 50-250 nm from mesenchymal stem cells, the method comprising the step of

providing a cell culture supernatant from the mesenchymal stem cells- conditioned cell culture medium, the cell culture supernatant comprising exosomes;

contacting the supernatant with polyethylene glycol having a molecular weight of 2,000-6,000 daltons to isolate and purify the exosomes; and

further purifying the isolated exosomes via the method of polyethylene glycol precipitation, wherein the polyethylene glycol has a molecular weight of 4,000-12,000 daltons.

2. The method according to claim 1, wherein the mesenchymal stem cells are derived from cardiac tissue, adipose tissue, neural tissue, insulin producing cells or umbilical cord tissue.

3. The method according to claim 2, wherein the mesenchymal stem cells are cultured in the cell culture medium at a dissolved carbon dioxide concentration of between 1-19%.

4. The method composition according to any one of claims 1 to 3, wherein the cell culture medium is selected from knockout DMEM or MesenCult™-hPL.

5. An exosome prepared from the method according to any one of claim 1 to 4 has a protein content of 0.5-100 pg.

6. An exosome prepared from the method according to any one of claim 1 to 4, the exosome is positive for at least one of the cellular exosome markers consisting of the group of CD9, CD63, CD81, TSG101, Annexins and HSP70.

7. An exosome prepared from the method according to any one of claim 1 to 4, the exosome is negative for at least one of the cellular exosome markers consisting of the group of GRP94 and Calnexin.

8. A pharmaceutical composition comprising the exosome according to any one of claim 5 to 7.