System for producing extracellular vesicles and method for producing extracellular vesicles

Patent metadata

Field	Value
Publication number	US-2025270492-A1
Application number	US-202318858354-A
Country	US
Kind code	A1
Filing date	Apr 20, 2023
Priority date	Apr 20, 2022
Publication date	Aug 28, 2025
Grant date	—

Abstract

A system for producing extracellular vesicles is provided. The system includes: a circulating culture medium type extracellular vesicle production unit that cultures cells with a circulating medium to produce a culture containing extracellular vesicles; and a separation unit that separates and obtains extracellular vesicles having a size of 200 nm or less, including exosomes, from any one or more stock solutions of the harvested culture and a cell disruption solution derived from the cultured cells. According to this, the production of target extracellular vesicles can be increased relative to the number of cells to be cultured, and the loss of target extracellular vesicles can be reduced from the produced culture and cultured cells, such that the target extracellular vesicles can be separated and obtained in excellent yield.

First claim

What is claimed is:

1 . An extracellular vesicle production system, comprising:
a medium circulation-type extracellular vesicle (EV) production unit for producing a culture containing EVs by culturing cells in a circulating medium; and a separation unit for separating and obtaining the EVs with a size of 200 nm or less, comprising exosomes, from at least one stock solution selected from a recovered culture and a cell lysate derived from cultured cells.

2 . The EV production system of claim 1 , wherein the medium circulation-type EV production unit comprises a cell culture unit, a medium storage unit, a circulation pump provided, wherein a medium stored in the medium storage unit circulates between the cell culture unit and the medium storage unit, and a gas supply unit supplying a gas to the medium provided to the cell culture unit.

3 . The EV production system of claim 2 , wherein the cell culture unit comprises a culture hosing with a culture space and a plurality of plate-shaped cell culture supports spaced 5.0 mm or less apart in a plurality of stages inside the culture housing.

4 . (canceled)

5 . The EV production system of claim 1 , wherein the separation unit comprises a first filter unit for separating and obtaining first particles with the size of 200 nm or less from the stock solution, a second filter unit for separating and removing second particles with a size of less than 50 nm from separated first particles, and a recovery unit for storing separated the EVs with a size of 50 to 200 nm.

6 . The EV production system of claim 5 , wherein the separation unit further comprises a third filter unit for separating third particles with a size exceeding 200 nm between the second filter unit and the recovery unit.

7 . The EV production system of claim 5 , wherein the second filter unit is configured to employ one or more of tangential flow filtration and size exclusion chromatography.

8 . The EV production system of claim 5 , wherein the separation unit further comprises a cell lysis unit for lysing the cultured cells by applying a physical force to the cultured cells or a culture containing the cultured cells, wherein the cultured cells and the culture containing the cultured cells are supplied from the medium circulation-type EV production unit, and the cell lysate produced by the cell lysis unit is supplied to the first filter unit.

9 . The EV production system of claim 8 , wherein the cell lysis unit comprises an inlet, an outlet, at least three fluid channels, and a plurality of partitions disposed to block each fluid channel, wherein a lysis target is supplied through the inlet, the cell lysate is discharged through the outlet, the lysis target passes through the at least three fluid channels, the at least three fluid channels connect the inlet and the outlet and have a width of 1 mm or less, the plurality of partitions have a plurality of microchannels passing through the plurality of partitions to lyse cells in the lysis target, and the plurality of partitions comprise a first partition and a second partition disposed closer to the outlet than the first partition, and a width of each microchannel formed in the first partition is 10 to 20 μm, and a width of each microchannel formed in the second partition is 1 to 5 μm.

10 . A method of producing extracellular vesicles (EVs), comprising:
(1) producing a culture comprising the EVs by culturing cells in a circulating medium; and (2) separating and obtaining the EVs with a size of 200 nm or less, comprising exosomes, from a stock solution comprising one or more selected from the culture and a cell lysate derived from cultured cells.

11 . The method of claim 10 , wherein the step (1) comprises a cell seeding step for supplying cells mixed in a medium to a cell culture unit, a cell culture step for producing the EVs by circulating the medium, wherein the medium discharged through ene a side of the cell culture unit is supplied back to the cell culture unit, and a step of obtaining a culture containing produced EVs.

12 . The method of claim 10 , wherein the medium is circulated at a rate of 20 to 100 mL/min, maintained at a pH of 7.5 to 8.0 with a CO 2 concentration of 4.5 to 6%, and the cells are cultured at 37 to 38° C.

13 . The method of claim 11 , wherein no bubbles are contained in the medium supplied to the cell culture unit.

14 . The method of claim 10 , wherein the cells are cultured on a plurality of plate-shaped cell culture supports, main surfaces of the plurality of plate-shaped cell culture supports face other at a predetermined interval and the main surfaces are arranged perpendicular to a ground, and the medium is circulated to flow through a space between the plurality of plate-shaped cell culture supports in a bottom-to-top direction from the ground.

15 . The method of claim 11 , wherein the cell culture unit comprises a plurality of plate-shaped cell culture supports, main surfaces of the plurality of plate-shaped cell culture supports face other at a predetermined interval,
the cell seeding step includes orienting the cell culture unit so that the main surfaces of the plurality of plate-shaped cell culture supports are substantially parallel to a ground to settle the cells onto the plurality of plate-shaped cell culture supports, and the cell culture step includes culturing the cells by orienting the cell culture unit so that the main surfaces of the plurality of plate-shaped cell culture supports are substantially perpendicular to the ground.

16 . The method of claim 11 , wherein in the cell culture step, the cells are proliferated to occupy 80 to 90% of a total effective cell culture area in the cell culture unit, and then a medium not containing foreign EVs is circulated.

17 . The method of claim 10 , wherein the step (2) comprises a first filtration step for separating a first filtrate comprising first particles with the size of 200 nm or less, comprising the EVs, from the stock solution; and
a second filtration step for separating and removing second particles with a size of less than 50 nm from the first filtrate.

18 . The method of claim 17 , wherein the second filtration step is performed on the first filtrate diluted 3.0-fold to 15.0-fold with a buffer.

19 . The method of claim 17 , wherein the first filtration step is performed by multi-stage filtration of the stock solution to produce a filtrate containing smaller particles at each stage, or performed with the stock solution diluted 3.0-fold to 15.0-fold with a buffer.

20 . The method of claim 17 , further comprising:
a third filtration step for separating and removing third particles with a size exceeding 200 nm from a second filtrate undergone the second filtration step.

21 . (canceled)

22 . A medium composition for cell culture, comprising particles with a size of 200 nm or less, comprising extracellular vesicles, as a cell culture additive.

Assignees

• Amogreentech Co Ltd

Inventors

• Park Hee Sung
• Lee Su-Yeon
• Seo In-Yong
• Kim Ji Young

Classifications

• C12M29/18

  External loop; Means for reintroduction of fermented biomass or liquid percolate (loop type reactors for chemical or physical processes B01J19/2435) · CPC title

• C12M47/10

  Separation or concentration of fermentation products (bioreactors combined with means for distillation or extraction of liquid fuel C12M43/02) · CPC title

• C12M47/06

  Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material (lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003) · CPC title

• C12M29/04

  Filters; Permeable or porous membranes or plates, e.g. dialysis · CPC title

• C12M33/14 Primary

  with filters, sieves or membranes · CPC title