CELL EXTRUDER AND CELL EXTRUSION METHOD

Abstract

A cell extruder of the present invention comprises: a pressure vessel in which a sample is dispensed; a regulator for adjusting a set pressure of nitrogen gas injected into the pressure vessel and then maintaining a constant pressure; an input valve for opening and closing the injection of nitrogen gas into the pressure vessel; an exhaust valve for removing the internal pressure of the pressure vessel; a filter holder which is provided with a membrane filter soaked with a reagent, so that the sample in the pressure vessel is fed through by the nitrogen gas and crushed into extracellular vesicles; and a collection container in which the extruded sample is stored. According to the present invention, pressure can be freely adjusted by the regulator. That is, the flow rate of the membrane according to pressure can be controlled, thus allowing the validation of a biopharmaceutical production method using an extracellular vesicle extruder. In addition, during cell extrusion, a sintered disc can be removed and cells can be extruded by using only a membrane. Vesicles can be produced within a fine pressure adjustment range (at a fine low pressure at which the membrane is not torn) in which no sintered disc is required.

Claims

1. A cell extruder comprising: a pressure vessel in which a sample is dispensed; a regulator for adjusting a set pressure of gas injected into the pressure vessel and then maintaining a constant pressure; an input valve for opening and closing the injection of gas into the pressure vessel; an exhaust valve for removing an internal pressure of the pressure vessel; a filter holder which is provided with a membrane filter, so that a sample in the pressure vessel is fed through by the gas and crushed into small pieces; and a collection container in which the extruded sample is stored.

2. The cell extruder of claim 1, further comprising: a pressure gauge for measuring the pressure of the pressure vessel.

3. The cell extruder of claim 2, further comprising: a diaphragm valve that opens a flow path to the filter holder only when the gas pressure acts on a diaphragm to reach the set pressure.

4. The cell extruder of claim 3, wherein the pressure vessel, the regulator, the input valve, the exhaust valve, the filter holder, the collection container, the pressure gauge, and the diaphragm valve are disassembled and assembled from and with adjacent components by clamps.

5. The cell extruder of claim 1, wherein the membrane filters are provided in an order of decreasing a pore size.

6. The cell extruder of claim 5, wherein the membrane filters are provided at the same time or sequentially replaced in the order of decreasing the size.

7. The cell extruder of claim 1, wherein the pressure vessel and the filter holder are connected to each other by a pipe provided with a nozzle.

8. The cell extruder of claim 7, wherein a diameter ratio of the nozzle and the filter holder is 1:3 to 1:30.

9. The cell extruder of claim 1, wherein the gas is at least one selected from the group consisting of nitrogen gas, compressed air, carbon dioxide, and inert gas.

10. A manufacturing method of cell-derived extracellular vesicles comprising applying the cell extruder of claim 1 to cells.

11. A manufacturing method of extracellular vesicles comprising: 1) supplying a sample containing cells to the cell extruder of claim 1; 2) extruding the sample by driving the cell extruder of step 1); and 3) obtaining the sample extruded in step 2).

12. The manufacturing method of extracellular vesicles of claim 11, wherein step 2) includes i) dispensing the sample to a pressure vessel; ii) supplying gas to the pressure vessel by opening an input valve; and iii) passing the sample through a membrane filter in a filter holder from the pressure vessel through a nozzle in a pipe.

13. The manufacturing method of extracellular vesicles of claim 12, wherein the gas is supplied at a pressure of 20 to 100 psi.

14. The manufacturing method of extracellular vesicles of claim 12, wherein step iii) includes passing the sample through the membrane filters sequentially in an order of decreasing a pore size.

15. The manufacturing method of extracellular vesicles of claim 14, wherein the membrane filters are provided at the same time or replaced sequentially in the order of decreasing the size.

16. (canceled)

Description

DESCRIPTION OF DRAWINGS

[0043] FIG. 1 is an exploded view of components of a cell extruder according to the present invention.

[0044] FIG. 2 is a schematic diagram illustrating an assembling state of FIG. 1.

BEST MODE OF THE INVENTION

[0045] Hereinafter, a preferred exemplary embodiment of the present invention will be described in detail with reference to the accompanying drawings.

[0046] FIG. 1 is an exploded view of components of a cell extruder according to the present invention and FIG. 2 is a schematic diagram illustrating an assembling state of FIG. 1.

[0047] As illustrated in FIGS. 1 and 2, a cell extruder A according to the present invention is configured by a pressure vessel 100 in which a sample is dispensed; a regulator 200 for adjusting a set pressure of gas injected into the pressure vessel 100 and then maintaining a constant pressure; an input valve 300 for opening and closing the injection of gas into the pressure vessel 100; an exhaust valve 400 for removing the internal pressure of the pressure vessel; a filter holder 500 which is provided with a membrane filter soaked with a reagent, so that a sample in the pressure vessel 100 is fed through by the gas and crushed into extracellular vesicles; and a collection container 600 in which the extruded sample is stored.

[0048] The pressure vessel 100 is clamped to a stand 150 and then maintained in an upright form, and includes an input line 110 to which gas is inputted, an exhaust line 120 for discharging the internal pressure of the pressure vessel 100, an injection line 130 for injecting the sample into the pressure vessel 100, and a pressure measuring line 140 for measuring the internal pressure of the pressure vessel 100.

[0049] The regulator 200 is to maintain the constant pressure after adjusting the input pressure to the set pressure. Specifically, the regulator 200 is configured to obtain an extrudate of uniform quality by injecting the sample into the pressure vessel 100 while maintaining the constant pressure after adjusting the gas of a set pressure or higher to the set pressure and applying uniform pressure.

[0050] The input valve 300 is connected to the input line 110, and the exhaust valve 300 is a well-known opening/closing valve connected to the exhaust line 120 to open and close the flow path. The detailed description of the structure thereof will be omitted and the operation thereof will be described in an operation description to be described below.

[0051] The filter holder 500 has an open/close structure and is formed to accommodate a membrane filter therein. At this time, the membrane filter crushes the sample fed through the gas from the pressure vessel 100 into the extracellular vesicles.

[0052] The gas is supplied to the filter holder 500 through a pipe 510 provided with a nozzle 511, and a diameter ratio between the nozzle 511 and the filter holder 500 is 1:3 to 1:30.

[0053] The membrane filters may be provided in the order of decreasing the pore size, and may be simultaneously provided in the cell extruder or sequentially replaced.

[0054] The collection container 600 has a container shape with a part opened, and stores the extruded sample.

[0055] In addition, a pressure gauge 700 for measuring the pressure of the pressure vessel 100 is further provided to visually display whether the internal pressure of the pressure vessel maintains the set pressure. In this case, the pressure gauge 700 may be added with a function of generating an alarm when the pressure is greater than or less than the set pressure.

[0056] In addition, a diaphragm valve 800 that opens the flow path to the filter holder 500 only when the gas pressure acts on a diaphragm to reach the set pressure is further provided. That is, when the pressure of the gas feeding the sample does not reach the set pressure, the sample is restricted from moving to the filter holder 500.

[0057] The gas may be at least one selected from the group consisting of nitrogen gas, compressed air, carbon dioxide, and inert gas.

[0058] In addition, an exhaust filter 900 is provided at a discharge end of the exhaust valve 400.

[0059] Meanwhile, the pressure vessel 100, the regulator 200, the input valve 300, the exhaust valve 400, the filter holder 500, the collection container 600, the pressure gauge 700, the diaphragm valve 800, and the exhaust filter 900 may be disassembled and assembled from and with adjacent components by clamps 101 to facilitate cleaning.

[0060] In addition, the overall size and volume of the cell extruder A of the present invention may be appropriately adjusted by the clamps 101 to be assembled, so that it is easy to be handled in the biological safety cabinet having a limited space.

[0061] In addition, the membrane filter installed into the filter holder 500 is provided in the filter holder 500 in the order of decreasing the pore size, and the membrane filters may be provided in the cell extruder at the same time or may be sequentially replaced. For example, the pore size of the membrane filter may be 10 μm, 8 μm, and 5 μm.

[0062] The operation of the cell extruder according to the present invention will be described to be divided into preparing, setting, operating, and cleaning with reference to FIGS. 1 and 2.

[0063] Preparing

[0064] 1) Cell extruder of the present invention provided with 47 mm disk holder

[0065] 2) Sample: Adipose derived stem cell (5E+6 cells/ml, total 20 ml)

[0066] 3) Reagent: PBS (PHOSPHATE BUFFERED SALINE)

[0067] 4) Consumables required: Membrane filter (whatman PC membrane filter 10, 8, 5 μm), pipette, petri dish

[0068] Setting

[0069] 1) Prepare the cell extruder A of the present invention of FIGS. 1 and 2 sterilized with an autoclave.

[0070] 2) Fill a petri dish with a small amount of reagent and soak the membrane filter with a 10 μm pore size.

[0071] 3) Insert and then lock the membrane filter soaked with the reagent into the filter holder 500.

[0072] 4) Sequentially mount the cell extruder A of the present invention on the stand 150.

[0073] 5) Mount a sample container on an end of the filter holder 500.

[0074] 6) Turn on the pressure gauge 700.

[0075] 7) Connect a tube supplying gas to the regulator 200. At this time, the input valve 300 is in a closed state.

[0076] 8) Supply the gas to the regulator 200 to maintain the gas at a set pressure of 50 psi.

[0077] Operating

[0078] 1) Open the injection line 130 of the pressure vessel 100 and dispense 20 ml of the reagent using a pipette.

[0079] 2) Close the injection line 130 and close the exhaust valve 400.

[0080] 3) Open the input valve 300 to supply the gas to the pressure vessel 100.

[0081] 4) Validate whether the reagent comes out at a constant rate (validation method: requiring 3 seconds based on 20 ml of PBS).

[0082] 5) Remove the internal pressure of the cell extruder A of the present invention including the pressure vessel 100 by closing the input valve 300 and opening the exhaust valve 400 when the conditions of step 4) above are satisfied.

[0083] 6) Open the injection line 130 of the pressure vessel 100.

[0084] 7) Resuspend a sample prepared in a total of 20 ml at a concentration of 5E+6/ml using a pipette and then dispense the sample into the pressure vessel 100 through the injection line 130.

[0085] 8) Close the injection line 130 and close the exhaust valve 400.

[0086] 9) Open the input valve 300 to supply the gas to the pressure vessel 100.

[0087] 10) Validate whether the sample is extruded and comes out.

[0088] 11) Remove the internal pressure of the cell extruder A of the present invention including the pressure vessel 100 by closing the input valve 130 and opening the exhaust valve 400.

[0089] 12) Open the injection line 130 of the pressure vessel 100 again.

[0090] 13) Dispense the extruded sample into the pressure vessel 100 through the injection line 130 using a pipette again.

[0091] 14) Repeat steps 6) to 13) twice to perform the extruding three times per membrane filter of one size.

[0092] 15) Separate the filter holder 500.

[0093] 16) Remove a membrane filter of 10 μm previously installed in the filter holder 500.

[0094] 17) Soak a membrane filter having a next size of 8 μm in the reagent in the petri dish.

[0095] 18) Insert and then lock the membrane filter of 8 μm into the filter holder 500.

[0096] 19) Reconnect the filter holder 500 to the cell extruder of the present invention.

[0097] 20) Repeat steps 6) to 14) above.

[0098] 21) Soak a membrane filter having a next size of 5 μm in the reagent in the petri dish.

[0099] 22) Repeat steps 6) to 14) above.

[0100] 23) Filter the sample that has been extruded using a 0.2 μm steri-cup.

[0101] 24) Store a filtered CDV sample collected in a 50 ml conical tube at 4° C. (short-term storage) or −80° C. (long-term storage) for the next step (purification or gUC).

[0102] Cleaning

[0103] 1) Separate the cell extruder A of the present invention from the stand 150 and then disassemble all clamps 101, and clean the entire configuration by immersing in 0.2 M NaOH for 30 minutes, followed by rinsing with water. At this time, the exhaust filter 900 is not cleaned.

[0104] 2) Reconnect and assemble the cleaned cell extruder A of the present invention through the clamps 101.

[0105] 3) Wrap the entire cell extruder A of the present invention with aluminum foil.

[0106] 4) Sterilize the cell extruder A through an autoclave.

[0107] 5) Put the sterilized extruder in a bench to perform UV sterilization.

[0108] As described above, although the preferred exemplary embodiments of the present invention have been described in detail, the technical scope of the present invention is not limited to the above-described exemplary embodiments and should be interpreted according to the appended claims. At this time, those skilled in the art will understand that many modifications and variations can be made without departing from the scope of the present invention.