METHOD OF PRODUCING PLANT-DERIVED EXOSOMES

Abstract

A method of producing plant-derived exosomes from plant tissue culture based cell suspension cultures is provided. The method includes: obtaining the plant tissue culture based cell suspension culture; mixing the the plant tissue culture based cell suspension culture with an isolation solution; centrifuging to obtain a supernatant and an infranatant; and obtaining the plant-derived exosomes from the infranatant. The objective of the present invention is to produce homogenous plant exosomes with high volume and purity by making use of the advantages of the plant suspension culture to be used for purposes such as therapeutics and drug carriers.

Claims

1. A method of producing plant-derived exosomes from a plant tissue culture based cell suspension culture comprising the following steps: obtaining the plant tissue culture based cell suspension culture, making a regularly subcultured callus culture from plants by a wounding method, ready to be transferred to a liquid culture within 2-3 weeks after a subculturing, dividing the regularly subcultured callus culture into pieces of 1-5 mm and placing the pieces into Erlenmeyer flasks such that the Erlenmeyer flasks are 10-50% full, preparing a liquid culture medium in each of the Erlenmeyer flasks such that the liquid culture medium contains sucrose, 6-Benzylaminopurine, 1-Napthaleneacetic acid, and a Murashige & Skoog vitamin-containing salt mixture, maintaining the liquid culture medium continuously under light during growth and agitating the liquid culture medium at an agitation speed of 80-120 rpm at a temperature of 20-26° C., performing a sub-culture via a vacuum filtration system at intervals of 5-10 days, straining through a sterile steel sieve once every 3-5 subcultures, mixing the plant tissue culture based cell suspension culture with an isolation solution at a ratio of 1:1 by inverting 20 times to obtain a first resulting mixture, centrifuging the first resulting mixture at 1500 g for 10 minutes at +4° C., after a centrifugation process, obtaining two separated phases as a supernatant accounting for 90% and comrpising a protein and other cellular wastes and an infranatant accounting for 10% and comprising the plant-derived exosomes, pulling and discarding the supernatant, transferring the infranatant containing the plant-derived exosomes to a clean tube, obtaining a solution as a supernatant of an aqueous two-phase system obtained by diluting the isolation solution with water at a ratio of 1:1 and centrifuging at 1000×g for 10 minutes, adding the solution at the ratio of 1:1 to the infranatant containing the plant-derived exosomes to obtain a second resulting mixture and inverting the second resulting mixture 10 times, centrifuging the second resulting mixture at 12000-14000 g for 10 minutes at +4° C., upon a collection of a supernatant from the second resulting mixture, removing ethanol (EtOH) in the solution by an evaporator to obtian the plant-derived exosomes, storing the plant-derived exosomes as a final product.

2. The method of producing the plant-derived exosomes according to claim 1, wherein a plant tissue used is tobacco leaves.

3. The method of producing the plant-derived exosomes according to claim 1, wherein a plant tissue used is stevia leaves.

4. The method of producing the plant-derived exosomes according to claim 1, wherein the liquid culture medium in each of the Erlenmeyer flasks is prepared such that it will the liquid culture medium contains 20-30 g/L of the sucrose, 6-Benzylaminopurine, 1-3 mg/L, of the 1-Napthaleneacetic acid, 3.5-4.5 g/L of the Murashige & Skoog vitamin-containing salt mixture.

5. The method of producing the plant-derived exosomes according to claim 1, wherein the liquid culture medium in each of the Erlenmeyer flasks comprises 0.1-0.8 mg/L of the 6-Benzylaminopurine when tobacco leaves are used as a plant tissue.

6. The method of producing the plant-derived exosomes according to claim 1, wherein the liquid culture medium in each of the Erlenmeyer flasks comprises 1-4 mg/L of the 6-Benzylaminopurine when stevia leaves are used as a plant tissue.

7. The method of producing the plant-derived exosotnes according to claim 1, wherein the plant-derived exosotnes are the final product and aliquoted and stored at −80° C. for up to 12 months.

8. The method of producing the plant-derived exosomes according to claim 1, wherein the plant-derived exosomes are the final product and lyophilized and stored at +4° C. for up to 36 months in a powder form.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figures of the method of producing plant-derived exosomes of the present invention are described as follows:

[0016] FIG. 1 shows the microscopic images of the cultured tobacco cells.

[0017] FIG. 2 shows the microscopic images of the cultured stevia cells.

[0018] FIG. 3 shows the measurement of the distribution of the sizes of the exosomes obtained from stevia plant cell suspension culture via dynamic light scattering.

[0019] FIG. 4 shows the measurement of the distribution of the sizes of the exosomes obtained from stevia plant cell suspension culture via dynamic light scattering.

[0020] FIG. 5 shows the view of the morphologies and sizes of the exosomes obtained from tobacco plant from different plant cultures by SEM image.

[0021] FIG. 6 shows the view of the morphologies and sizes of the exosomes obtained from stevia plant from different plant cultures by SEM image.

[0022] FIG. 7 shows a graphical representation of the control group carried out for characterization of the exosomes obtained from tobacco cells by flow cytometry.

[0023] FIG. 8 shows a graphical representation of the measurement of the CD 9 proteins of the exosomes obtained from tobacco cells by flow cytometry.

[0024] FIG. 9 shows a graphical representation of the measurement of the CD 63 proteins of the exosomes obtained from tobacco cells by flow cytometry.

[0025] FIG. 10 shows a graphical representation of the measurement of the HSP70 proteins of the exosomes obtained from tobacco cells by flow cytometry.

[0026] FIG. 11 shows a graphical representation of the control group carried out for characterization of the exosomes obtained from stevia cells by flow cytometry.

[0027] FIG. 12 shows a graphical representation of the measurement of the CD9 proteins of the exosomes obtained from stevia cells by flow cytometry.

[0028] FIG. 13 shows a graphical representation of the measurement of the CD63 proteins of the exosomes obtained from stevia cells by flow cytometry.

[0029] FIG. 14 shows a graphical representation of the measurement of the IHSP70 proteins of the exosomes obtained from stevia cells by flow cytometry.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The present invention relates to a method of producing plant-derived exosomes from plant tissue culture-based cell suspension cultures comprising the following steps: [0031] Obtaining the plant cell suspension culture, [0032] a Making the regularly subcultured callus culture, whith is obtained from plants (preferably from tobacco leaves or stevia leaves) by wounding method, ready to be transferred to liquid culture within 2-3 weeks after subculturing, [0033] Dividing the callus culture into small pieces of 1-5 mm and placing the pieces into Erlenmeyer flasks such that the flasks will be 10-50% full, [0034] Preparing the liquid culture medium in the Erlenmeyer flask such that it will contain 20-30 g/L sucrose, (in case tobacco leaves are used 0.1-0.8 mg/L or in case stevia leaves are used 1-4 mg/L) 6-Benzylaminopurine, 1-3 mg/L 1-Napthaleneacetic acid, 3.5-4.5 g/L Murashige & Skoog vitamin-containing salt mixture, [0035] Maintaining the liquid culture continuously under light during growth and agitating at an agitation speed of 80-120 rpm at a temperature of 20-26° C., [0036] Performing sub-culture via vacuum filtration system at intervals of 5-10 days, [0037] Straining through a sterile steel sieve once every 3-5 subcultures, [0038] Mixing the plant culture media with the isolation solution containing 2-1% polyethylene glycol with a molecular weight of 25-45 kDa and 1-2% dextran with a molecular weight of 450-650 kDa at a ratio of 1:1 by inverting 20 times, [0039] Centrifuging at 1500 g for 10 minutes at +4° C., [0040] After the centrifugation process, obtaining two separated phases as the supernatant comprising 90% of the total and containing protein and other cellular wastes, and the infranatant comprising 10% and where exosomes are collected, [0041] Carefully pulling and discarding the supernatant, [0042] Transferring the infranatant containing exosomes to a clean tube, [0043] Obtaining Solution C as the supernatant of the aqueous two-phase system obtained by diluting the isolation solution with water at a ratio of 1:1 and centrifuging at 1000 x g for 10 minutes, [0044] Adding solution C at a ratio of 1:1 to the said infranatant containing exosome and inverting it 10 times, [0045] Centrifuging the mixture at 12000-14000 g for 10 minutes at +4° C., [0046] Upon collection of the supernatant, removing the ethanol (EtOH) in solution C by means of an evaporator, [0047] Storing the obtained exosomes as the final product (at −80° C. for up to 12 months upon aliquoting, or at +4° C. for up to 36 months in powder form upon lyophilizing).

[0048] The present invention relates to a method of producing plant-derived exosomes from plant tissue culture-based cell suspension cultures. In the said method, firstly, tobacco and stevia cell suspension cultures are created, then plant exosomes are obtained by using the said cell suspension cultures.

[0049] Within the scope of the invention, the culture medium is ensured to be treated with sugar, salt, vitamins and hormones. In this process, 6-Benzylaminopurine is preferred as the hormone (6-Benzylaminopurine; benzyl adenine, BAP or BA is a first-generation synthetic cytokinin that promotes plant growth and development responses, setting blossoms and stimulating fruit richness by stimulating cell division. Callus tissue, which is regularly subcultured and obtained from plants (preferably tobacco leaves or stevia leaves) by wounding method, is formed by stimulating the said leaf tissues with suitable hormone concentrations. Callus culture is prepared in which the properties of the obtained callus tissue are continuously preserved with the help of certain hormones. Tobacco and Stevia are different plant species, and they need to be regularly stimulated with certain hormones to protect the callus culture. These hormones vary between species. Sugar, salt and vitamins can also vary, but the same sugar and salt ratios have been found to be suitable for Tobacco and Stevia. These hormones vary between species. Sugar, salt and vitamins can also vary. Murashige & Skoog salt mixture containing vitamins [14], which is widely used in the state of the art, is considered as the vitamin-salt mixture used herein. Said Murashige & Skoog mixture, named after the researchers who invented it, is a medium composition frequently used in plant tissue culture. The “vitamin salt mixture” referred to herein is a liquid-liquid medium obtained using Murashige & Skoog powder. Amount of Murashige & Skoog used is prepared such that “Murashige & Skoog vitamin-containing salt mixture comprises [10] 3,5-4,5 g/L [11].

[0050] It has been determined in the studies conducted in present time that by isolating the exosomes obtained from plants from the medium used in plant cell culture, very important advantages have emerged in terms of the homogeneity of the exosomes, amount of production and genetic applications. The problem of not being able to obtain a homogeneous exosome culture, which is one of the most important obstacles in studying the bioactivity of plant-derived exosomes, has been solved within the scope of the invention. In the method of the invention, exosomes are secreted into the medium in the plant cell suspension culture by a single cell type and the said cells are grown under controlled conditions. Thus, it is possible over time to minimize the vesicular structure and content differences that will occur during the production of exosomes required in experimental studies carried out. Another advantage of this is that the medium used in plant tissue culture contains much less contamination than a fruit extract to be used for exosome purification. Accordingly, significant advantages are obtained in exosome isolation in terms of both time and efficiency.

[0051] Within the scope of the invention, a process is provided for the purification of exosomes from plants, which is generally independent from the growth conditions and areas required for plants. In the field of the invention, in fruit-based studies, the problems such as the facts that the plants have specific dates when they characteristically yield products and that the amount of area required to obtain the desired amount of exosomes is too large are overcome with the method of the present invention. By using bioreactors for plant cultures, the exosomes of the cells intended to be studied in plants can be obtained independent from time and in very high amounts in more minimal areas.

[0052] Within the scope of the present invention, obtaining exosomes from the cell population in plant tissue culture enables to examine the responses of plant cells to environmental changes. Moreover, by incorporating a special protein into the vesicular structure in the regulation of plant-derived exosome cargoes, it will be possible to make the responses of genetic changes on plant cells cell-specific.

[0053] Within the scope of the invention, aliquoting or lyophilization processes are applied to preserve the exosome, which is the final product, for a long time. Aliquoting is for preventing exposure to a repetitive freeze-thaw process. Lyophilization provides a long-term stability at +4 degrees. These processes are used for the correct storage of our final product.

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