Roller bottle culture method for stem cells for mass production of extracellular vesicles

Abstract

This invention relates to a stem cell culture method using a roller bottle for improving the yield and quality of separation and purification of extracellular vesicles, and to a method for separating stem cell-derived extracellular vesicles. When exosomes or extracellular vesicles are separated from a culture medium obtained by culturing stem cells using the roller bottle method, the yield is greatly increased, and high-quality extracellular vesicles with high CD9 and CD81 expression can be obtained. As a result, stem cells can be cultured in large quantities using the stem cell culture method using the roller bottle of the present invention, and exosomes and extracellular vesicles can be separated and purified with high yield.

Claims

1. One step involves inoculating stem cells into a roller bottle and culturing them in cell culture medium; Two steps: replacing the cell culture medium with serum-free medium and culturing in serum-free medium; and A method for separating stem cell-derived extracellular vesicles for mass production of extracellular vesicles, comprising three steps: tangential-flow filtration (TFF) to separate and purify extracellular vesicles.

2. The method for separating stem cell-derived extracellular vesicles according to claim 1, wherein the stem cell culture is performed by rotating a roller bottle at a rotational speed of 0.25 rpm to 2.0 rpm.

3. Surface area cm² of the aforementioned roller bottle ２ A method for isolating stem cell-derived extracellular vesicles according to claim 1, comprising inoculating and culturing 1,000 to 5,000 stem cells per unit.

4. The surface area of ​​the aforementioned roller bottle is 700 cm². ２ ~1000cm ２ The method for separating stem cell-derived extracellular vesicles according to claim 1.

5. A method for separating stem cell-derived extracellular vesicles according to claim 1, wherein stem cells are cultured simultaneously using multiple roller bottles during a single culture cycle.

6. The method for isolating stem cell-derived extracellular vesicles according to claim 1, wherein the stem cells are embryonic stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

7. The method for isolating stem cell-derived extracellular vesicles according to claim 6, wherein the adult stem cells are adult stem cells of human or animal tissue origin, mesenchymal stem cells derived from human or animal tissue, or mesenchymal stem cells derived from induced pluripotent stem cells of human or animal tissue.

8. The method for isolating stem cell-derived extracellular vesicles according to claim 7, wherein the human or animal tissue is selected from the group consisting of brain, teeth, liver, ovaries, peripheral blood, hair follicles, heart, umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, and placenta.

9. The method for isolating stem cell-derived extracellular vesicles according to claim 7, wherein the adult stem cells of human or animal tissue origin are selected from the group consisting of mesenchymal stem cells, endoetiological stem cells, hair follicle stem cells, muscle stem cells, ovarian stem cells, cardiac stem cells, hematopoietic matrix cells, mammary gland stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory neural stem cells, adipose stem cells, and testicular stem cells.

10. The method for separating stem cell-derived extracellular vesicles according to claim 1, wherein culturing stem cells using the roller bottle increases the yield of separation and purification of extracellular vesicles.

11. The method for separating stem cell-derived extracellular vesicles according to claim 1, wherein the separated and purified extracellular vesicles have a diameter of 95% or more of 100 nm to 300 nm.

12. The separated and purified extracellular vesicles are 10 per unit volume of culture medium. １１ 1 to 10 １３ A method for isolating stem cell-derived extracellular vesicles according to claim 1, comprising individual extracellular vesicles.

13. The method for separating stem cell-derived extracellular vesicles according to claim 1, wherein the proliferation rate of stem cells using the culture method with the roller bottle is reduced by 20% to 40% compared to the culture method using a flask.

14. The method for separating stem cell-derived extracellular vesicles according to claim 1, wherein the separated and purified extracellular vesicles are more uniform in size, have an increased protein concentration or particle count per unit volume, exhibit increased expression of CD9 and CD81 proteins, or have an increased anti-inflammatory effect compared to a culture method using a flask.

15. A method for culturing stem cells for the mass production of extracellular vesicles, including the step of inoculating stem cells into roller bottles and culturing them.

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