KR20230036085A - Apparatus for manufacturing of cell-derived vesicles(CDVs) and manufacturing method thereusing - Google Patents

Abstract

The present invention relates to a device for preparing cell-derived vesicles and a preparation method using same, the device comprising: a first filter assembly and a second filter assembly, one side of which a culture solution supply line to which a cell culture solution is supplied and a gas supply line are connected to, and in which a filter is provided; a first extrusion line connected to the other side of the first filter assembly; a second extrusion line which is connected to the other side of the second filter assembly and communicates with the first extrusion line; and a harvesting line which communicates with the first extrusion line and the second extrusion line. The present invention has the effect of enabling cell-derived vesicles to be prepared stably, economically, and in large quantities through a simplified process.

Description

Apparatus for manufacturing of cell-derived vesicles (CDVs) and manufacturing method thereusing}

The present invention relates to an apparatus for preparing cell-derived endoplasmic reticulum and a manufacturing method using the same. More specifically, it relates to a cell-derived endoplasmic reticulum manufacturing apparatus and a manufacturing method using the same for preparing cell-derived endoplasmic reticulum by transferring fluid containing nucleated or non-nucleated cells into a depth filter using a pressure container.

Extracellular vesicles refer to various types of particles secreted from cells, and include exosomes derived from the endosomal pathway and microvesicles derived from the plasma membrane. microvesicles), etc.

Exosomes, which are spherical vesicles excreted by cells, contain various information such as protein and DNA of the mother cell, and are being used as biomarkers to actively develop cancer diagnostic markers and sensors.

In addition, microvesicles are a kind of organelles generally having a size of 0.03 to 1 μm, which are naturally separated from cell membranes and have a double phospholipid membrane form, and cytoplasm such as mRNA, DNA, and proteins. It contains my ingredients.

In recent years, studies on drug delivery systems using these extracellular vesicles have been actively conducted, and since a small amount of a drug can be encapsulated in a vesicle to deliver the drug to a specific area, the use of extracellular vesicles in various medical fields is increasing. there is. In particular, since microvesicles are directly released from cell membranes and retain antigens of parent cells as they are, the possibility of using such as vaccines is very high.

However, since the amount of extracellular vesicles that can be obtained from cells is very limited, a method for preparing extracellular vesicles to artificially obtain a large amount of them has recently been required.

Conventionally, a centrifugal separator can be used as a method for obtaining extracellular vesicular mimics, but the centrifugal separator is expensive and still has limitations in yields not meeting expectations.

Korean Patent Publication No. 10-2014-0118524 (2014.10.08.)

An object of the present invention is to provide an apparatus for preparing cell-derived endoplasmic reticulum capable of easily increasing the production scale and simplifying the production process in preparing cell-derived endoplasmic reticulum and a manufacturing method using the same in order to solve the above problems.

In order to achieve the above object, the present invention provides a first pressure vessel and a second pressure vessel to which a culture medium supply line and a gas supply line are connected to one side of which a cell culture medium is supplied; a first extrusion line connected to the other side of the first pressure vessel; a second extrusion line connected to the other side of the second pressure vessel and communicating with the first extrusion line; at least one filter disposed on the first extrusion line or the second extrusion line through which the cell culture medium passes; and a harvest line communicating with the first extrusion line and the second extrusion line.

At this time, the filter is characterized in that at least one disposed on the first extrusion line and the second extrusion line.

In addition, the filter is characterized in that a depth filter (depth filter).

On the other hand, the present invention is connected to the culture medium supply line and the gas supply line to which the cell culture medium is supplied to one side, the first filter assembly and the second filter assembly provided with a filter inside; a first extrusion line connected to the other side of the first filter assembly; a second extrusion line connected to the other side of the second filter assembly and communicating with the first extrusion line; and a harvest line communicating with the first extrusion line and the second extrusion line.

At this time, the first filter assembly and the second filter assembly have lower ends in which at least one mounting hole is formed; a cartridge filter detachably provided in the mounting hole; a middle portion formed to accommodate the cartridge filter therein and coupled to the lower end portion; and an upper end fastened to an upper end of the middle part to seal the inside together with the lower end and the middle part.

In addition, the first filter assembly and the second filter assembly may include a plurality of intermediate portions provided to be coupled to and detachable from each other, and an inner space may be expanded by coupling the plurality of intermediate portions to each other.

In addition, the first filter assembly and the second filter assembly may further include a pressure regulator connected to the gas supply line to mix gas and adjust pressure.

In addition, the first filter assembly and the second filter assembly are provided to be detachable from the mounting hole, characterized in that further comprising a cap that seals the mounting hole in a state inserted into the mounting hole.

On the other hand, the present invention is a method for producing cell-derived endoplasmic reticulum using the cell-derived endoplasmic reticulum manufacturing apparatus, a) a solution containing cells into one or more of the first pressure vessel and the second pressure vessel putting in; b) opening the gas supply line connected to the first pressure vessel and closing the gas supply line connected to the second pressure vessel to allow the solution to pass through the filter; and c) closing the gas supply line connected to the first pressure vessel and opening the gas supply line connected to the second pressure vessel so that the solution passes through the filter in the opposite direction in step b). It provides a method for preparing a cell-derived endoplasmic reticulum comprising the step.

In this case, the method may further include d) repeating steps b) and c) a predetermined number of times.

In addition, the solution is a cell culture medium, and the step a) is characterized by injecting the cell culture medium into at least one of the first pressure vessel and the second pressure vessel through the culture medium supply line.

At this time, between steps a) and b), opening the gas supply line connected to the pressure vessel into which the cell culture medium is introduced allows the cell culture medium to pass through the filter; Discharging the used medium in which cells do not remain through the harvest line; and injecting a solution for suspending the cells through the harvest line.

In addition, the solution is a cell suspension, and the step a) is characterized by injecting the cell suspension into one or more of the first pressure container and the second pressure container through the culture medium supply line.

On the other hand, the present invention is a method for producing cell-derived endoplasmic reticulum using the cell-derived endoplasmic reticulum manufacturing apparatus, a) inside any one or more filter assemblies of the first filter assembly and the second filter assembly through the culture medium supply line Injecting a solution containing cells into the furnace; b) the gas supply line connected to the first filter assembly is opened, and the gas supply line connected to the second filter assembly is closed so that the cell culture medium enters the cartridge inside the first filter assembly and the second filter assembly. passing through a filter; and c) closing the gas supply line connected to the first filter assembly and opening the gas supply line connected to the second filter assembly so that the cell culture medium flows in the opposite direction in step b) to the first filter assembly. and passing through the cartridge filter inside the second filter assembly.

In this case, the method may further include d) repeating steps b) and c) a predetermined number of times.

In addition, the solution is a cell culture medium, and step a) is characterized in that the cell culture medium is introduced into at least one of the first filter assembly and the second filter assembly through the culture medium supply line.

At this time, between the step a) and the step b), opening the gas supply line connected to the filter assembly into which the cell culture medium is introduced to allow the cell culture medium to pass through the filter; Discharging the used medium in which cells do not remain through the harvest line; and injecting a solution for suspending the cells through the harvest line.

In addition, the solution is a cell suspension, and step a) is characterized in that the cell suspension is introduced into at least one of the first filter assembly and the second filter assembly through the culture medium supply line.

In addition, filtration or extrusion of cells is performed in the first filter assembly and the second filter assembly, or filtration and extrusion of cells are performed simultaneously.

The present invention has the effect of stably, economically, and mass-producing cell-derived endoplasmic reticulum through a simplified process.

1 is a diagram schematically showing an apparatus for manufacturing cell-derived vesicles according to a first embodiment of the present invention.
2 is a diagram schematically illustrating an apparatus for manufacturing cell-derived vesicles according to a second embodiment of the present invention.
3 is a diagram schematically illustrating an apparatus for manufacturing cell-derived endoplasmic reticulum according to a third embodiment of the present invention.
4 is a diagram schematically illustrating an apparatus for manufacturing cell-derived vesicles according to a fourth embodiment of the present invention.
5 and 6 are exploded perspective views of the filter assembly.
7 is a diagram illustrating the results of productivity, particle concentration, particle size, PDI, protein concentration, and DNA concentration trend according to the number of extrusions when cells are harvested and extruded using the apparatus for producing cell-derived endoplasmic reticulum according to a second embodiment of the present invention it is a drawing
8 is a diagram of the productivity results of particles per cell according to various parameters.
9 is a diagram of the results of the average extrusion process time according to various parameters.
10 is a diagram of the results of productivity and average extrusion processing time according to the amount of PBS per cell and the temperature of PBS.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. First, in adding reference numerals to components of each drawing, it should be noted that the same components have the same numerals as much as possible even if they are displayed on different drawings. In addition, if it is determined that the subject matter of the present invention may be obscured, the detailed description thereof will be omitted. In addition, although preferred embodiments of the present invention will be described below, the technical spirit of the present invention is not limited or limited thereto and can be practiced by those skilled in the art, of course.

1 is a diagram schematically showing an apparatus for manufacturing cell-derived vesicles according to a first embodiment of the present invention.

The apparatus 10 for preparing cell-derived vesicles according to the first embodiment of the present invention includes a pressure vessel 100 and a filter 110.

Specifically, a culture medium supply line 102 through which cell culture medium is supplied is connected to one side of the pressure vessel 100, a gas supply line 104, and a vent line 106, and an extrusion line 112 is connected to the other side.

On the path of the extrusion line 112, at least one filter 110 through which the cell culture fluid discharged from the pressure vessel 100 passes is disposed, and at an end of the extrusion line 112, a harvest line communicating with the extrusion line 112 (114) is provided.

At this time, the filter 110 is preferably a depth filter.

In addition, valves 120 are installed in the culture medium supply line 102, the gas supply line 104, the vent line 106, the extrusion line 112, and the harvest line 114, respectively, to control the opening and closing of the corresponding lines or to supply Alternatively, the pressure of the discharged fluid may be adjusted.

Gas such as air or nitrogen is supplied from a separate gas tank (not shown) connected to the gas supply line 104 through the gas supply line 104 .

A cell harvesting method for separating spent media from a cell culture medium using the cell-derived endoplasmic reticulum manufacturing apparatus 10 according to the first embodiment of the present invention is as follows.

First, the cell culture medium is supplied into the pressure vessel 100 through the culture medium supply line 102 . Then, the gas supply line 104 connected to the gas tank is opened, and the vent line 106 is closed so that the cell culture solution passes through the filter 110 through the extrusion line 112 under a predetermined pressure, and the harvest line ( 114) to discharge the spent media in which the cells do not remain.

A method for preparing cell-derived endoplasmic reticulum using the cell-derived endoplasmic reticulum manufacturing apparatus 10 according to the first embodiment of the present invention is as follows.

In the cell harvesting method, the spent media is discharged and the following steps are additionally performed.

First, with the gas supply line 104 closed, a solution suitable for suspending cells, such as phosphate buffered saline (PBS), is injected through the harvest line 114 into the pressure container 100. form a cell suspension; Thereafter, the gas supply line 104 connected to the gas tank is opened and the vent line 106 is closed so that the cell suspension in the pressure container 100 passes through the filter 110 through the extrusion line 112 under a predetermined pressure. And, the final extrusion solution is obtained through the harvest line 114.

In this case, the final extrusion solution may include cell-derived endoplasmic reticulum.

As described above, in the process of preparing cell-derived endoplasmic reticulum using the cell-derived endoplasmic reticulum manufacturing apparatus 10 according to the first embodiment of the present invention, cell harvesting, cell suspension preparation, and extrusion are performed only within the cell-derived endoplasmic reticulum manufacturing apparatus. , the risk of contamination or deformation of cells exposed to the external environment is reduced, the process can be simplified, and there are advantages in improving the process speed.

Meanwhile, in the cell-derived endoplasmic reticulum manufacturing method, a cell suspension may be supplied into the pressure vessel 100 through the culture solution supply line 102 instead of the cell culture solution. In this case, the step of forming a cell suspension in the pressure container 100 by injecting a solution suitable for suspending cells through a cell harvesting method and the harvesting line 114 may be omitted. In addition, at this time, the cell suspension may be a solution obtained by harvesting cells from a cell culture medium through centrifugation and resuspending cells in a solution suitable for resuspending cells such as PBS.

2 is a diagram schematically illustrating an apparatus for manufacturing cell-derived vesicles according to a second embodiment of the present invention.

The cell-derived endoplasmic reticulum manufacturing apparatus 20 according to the second embodiment of the present invention has a structure in which the cell-derived endoplasmic reticulum manufacturing apparatus 10 according to the first embodiment is symmetrically connected.

Specifically, in the cell-derived endoplasmic reticulum manufacturing apparatus 20 according to the second embodiment, the culture medium supply lines 102 and 102' through which the cell culture medium is supplied, the gas supply lines 104 and 104', and the vent lines 106 and 106' are connected to one side. The first pressure vessel 100 and the second pressure vessel 100 ', the first extrusion line 112 connected to the other side of the first pressure vessel 100, connected to the other side of the second pressure vessel 100', A second extrusion line 112' communicating with the first extrusion line 112, and filters 110 and 110' disposed on the first extrusion line 112 and the second extrusion line 112', respectively, through which the cell culture medium passes. , and a harvest line 114 communicating with the first extrusion line 110 and the second extrusion line 112 '.

In addition, valves 120 are installed in the culture medium supply lines 102 and 102', the gas supply lines 104 and 104', the vent lines 106 and 106', the extrusion lines 112 and 112', and the harvest line 114, respectively, to open and close the corresponding lines. can be adjusted or the pressure of the fluid supplied or discharged can be adjusted.

At this time, the gas supply lines 104 and 104' communicate with each other and can be connected to one gas tank (not shown), and the flow direction and pressure of the supplied gas can be adjusted by adjusting the opening and closing of the valve 120. there is.

In addition, the filters 110 and 110' are preferably depth filters, but the type of filter is not limited thereto, and any type of filter may be used as long as it is applicable to the production of cell-derived endoplasmic reticulum, such as a membrane filter. free

3 is a diagram schematically illustrating an apparatus for manufacturing cell-derived endoplasmic reticulum according to a third embodiment of the present invention.

Compared to the cell-derived endoplasmic reticulum manufacturing apparatus 20 according to the second embodiment, the cell-derived endoplasmic reticulum manufacturing apparatus 30 according to the third embodiment of the present invention has only one filter 110 except that it is provided. Other configurations are the same as those of the cell-derived endoplasmic reticulum manufacturing apparatus 20 of the second embodiment.

Although not specifically shown, the cell-derived endoplasmic reticulum manufacturing apparatuses 20 and 30 according to the second and third embodiments of the present invention may be formed in a structure in which three or more pressure vessels 100 are connected in parallel.

In this case, the added pressure vessel 100 has a gas supply line 104 and an extrusion line 112 connected to the culture medium supply line 102, the vent line 106, the gas tank and the harvest line 114, respectively, as described above. ) is installed.

In addition, a filter 110 may be disposed on the added extrusion line 112 as needed.

A method for preparing cell-derived endoplasmic reticulum using the cell-derived endoplasmic reticulum manufacturing apparatus 20 or 30 according to the second or third embodiment of the present invention is as follows.

First, the cell culture medium is introduced into one or more of the first pressure vessel 100 and the second pressure vessel 100' through the culture medium supply lines 102 and 102'.

Thereafter, the gas supply lines 104 and 104' connected to the gas tank are opened so that the cell culture medium passes through the filters 110 and 110' through the extrusion lines 112 and 112' under a predetermined pressure, and the cells are harvested through the harvest line 114. Drain any remaining spent media. Next, with the gas supply lines 104 and 104' closed, a solution suitable for suspending cells, such as phosphate buffered saline (PBS), is injected through the harvest line 114 into a pressure container ( 100,100') to form a cell suspension.

Subsequently, the gas supply line 104 connected to the first pressure vessel 100 is opened and the vent line 106 is closed. In addition, the gas supply line 104' provided in the second pressure vessel 100' is closed and the vent line 106' is opened so that the cell suspension in the first pressure vessel 100 flows through the first extrusion line 112. After passing through the filters 110 and 110', it is moved to the second pressure container 100'.

Then, contrary to the previous step, the gas supply line 104 connected to the first pressure container 100 is closed and the vent line 106 is opened. In addition, the gas supply line 104' provided in the second pressure vessel 100' is opened and the vent line 106' is closed so that the cell suspension contained in the second pressure vessel 100' is discharged from the previous step. It flows in the opposite direction, that is, in the direction from the second pressure vessel 100' to the first pressure vessel 100, and passes through the filters 110 and 110'.

As described above, the process of alternately opening and closing the gas supply lines 104 and 104' is repeated a predetermined number of times to allow the cell suspension to pass through the filters 110 and 110' while reciprocating a plurality of times, thereby increasing the production efficiency of cell-derived endoplasmic reticulum.

After allowing the cell suspension to pass through the filters 110 and 110' repeatedly a predetermined number of times, the harvest line 114 is opened to obtain a final extrusion solution.

In this case, the final extrusion solution may include cell-derived endoplasmic reticulum.

On the other hand, in the above manufacturing method, when cell-derived endoplasmic reticulum is prepared using the cell-derived endoplasmic reticulum manufacturing apparatus 30 according to the third embodiment, in the step of injecting the cell culture medium into the pressure vessel, the cell culture medium is subjected to the first pressure Among the vessel 100 and the second pressure vessel 100', it may be put into a pressure vessel adjacent to the filter 110.

As described above, in the process of preparing cell-derived endoplasmic reticulum using the cell-derived endoplasmic reticulum manufacturing apparatus 20 or 30 according to the second or third embodiment of the present invention, cell harvesting and extrusion are carried out only within the cell-derived endoplasmic reticulum manufacturing apparatus. Since this is done, there are advantages in that the risk of contamination or transformation of cells exposed to the external environment is reduced, the process can be simplified, and the process speed is improved.

Meanwhile, in the cell-derived endoplasmic reticulum manufacturing method, instead of the cell culture medium, the cell suspension may be supplied into the pressure vessels 100 and 100' through the culture medium supply lines 102 and 102'. At this time, the spent media in which cells do not remain are discharged through the harvest line 114 and a solution suitable for suspending the cells is injected into the pressure vessels 100 and 100' through the harvest line 114. The step of forming a cell suspension may be omitted. In addition, at this time, the cell suspension may be a solution obtained by harvesting cells from a cell culture medium through centrifugation and resuspending cells in a solution suitable for resuspending cells such as PBS.

4 is a schematic view of an apparatus for manufacturing cell-derived vesicles according to a fourth embodiment of the present invention, and FIGS. 5 and 6 are exploded perspective views of a filter assembly.

In the cell-derived endoplasmic reticulum manufacturing apparatus 40 according to the fourth embodiment of the present invention, the culture medium supply lines 102 and 102 'to which the cell culture medium is supplied, the gas supply lines 104 and 104', and the vent lines 106 and 106' are connected to one side, , The first filter assembly 200 and the second filter assembly 200 'in which the filter 220 is provided, and the first extrusion line 112 connected to the other side of the first filter assembly 200, the second A second extrusion line 112 'connected to the other side of the filter assembly 200' and communicating with the first extrusion line 112, and communicating with the first extrusion line 112 and the second extrusion line 112' It includes a harvest line 114.

In addition, each line (102, 102', 104, 104', 106, 106', 112, 112', 114) is provided with a valve 120, and the connection relationship thereof is the cell-derived endoplasmic reticulum manufacturing apparatus (20, 30) according to the second and third embodiments. ) is configured in the same way as

However, in the cell-derived endoplasmic reticulum manufacturing apparatus 40 according to the fourth embodiment, the filter 220 is not separately installed on the extrusion lines 112 and 112', but is provided in the filter assemblies 200 and 200'. There is a difference from the devices 20 and 30 of the third embodiment.

Specifically, the filter assemblies 200 and 200' include a lower end 210 in which at least one mounting hole 212 is formed, a cartridge filter 220 detachably provided in the mounting hole 212, and a cartridge filter 220 therein. Equipped with a middle part 230 formed to be accommodated and coupled to the lower end 210, and an upper end 240 that is fastened to the upper end of the middle part 230 to seal the lower end 210 and the middle part 230 and the inside. do.

Here, the lower part 210, the middle part 230, and the upper part 240 serve as a pressure vessel in which the inside is sealed in a state of being coupled to each other, and the cell culture medium supplied therein is filtered through the cartridge filter 220 or is extruded

At this time, the extrusion lines 112 and 112' are connected to the lower end 210, and the culture medium supply lines 102 and 102', the gas supply lines 104 and 104', and the vent lines 106 and 106' are connected to the upper end 240.

In addition, as shown in FIG. 6 , the filter assemblies 200 and 200 ′ may include a plurality of intermediate parts 230 provided to be coupled to and detachable from each other. The inner space of the filter assemblies 200 and 200' can be expanded by coupling the plurality of intermediate parts 230 to each other, and through this method, the extrusion scale and manufacturing capacity can be easily adjusted as needed.

In general, the maximum capacity of the pressure vessel (100, 100') is 2L and the applicable filter area is 0.026m ² ~ 0.1m ² , whereas the capacity of the filter assembly (200, 200') is 5L ~ 30L and the applicable filter area is 0.026m 2 ~ 0.1m 2 is 0.45 m ² ~ 2.7 m ² , and the maximum capacity of the filter assemblies 200 and 200' depends on the increase in the quantity of the middle part 230 or the expansion of the lower part 210, and the applicable filter area is the mounting hole 212 It has the advantage of increasing productivity in that it can be additionally increased according to facility expansion.

On the other hand, the cartridge filter 220 may be mounted as many as the number of mounting holes 212 formed in the mounting hole 212 formed at the lower end 210, but, if necessary, a smaller number than the number of mounting holes 212 It is also possible that the cartridge filter 220 is mounted.

In this case, in order to ensure smooth extrusion performance of the cell culture medium, it is preferable to close the idle mounting hole 212 on which the cartridge filter 220 is not mounted. A cap 214 for closing may be additionally provided.

The cap 214 is detachably provided in the mounting hole 212 and is formed to seal the mounting hole 212 while being inserted into the mounting hole 212 .

In addition, the filter assemblies 200 and 200' are connected to the gas supply lines 104 and 104' and may further include a pressure regulator for gas mixing and pressure control.

Although not specifically shown, the cell-derived endoplasmic reticulum manufacturing apparatus 40 according to the fourth embodiment of the present invention may be formed in a structure in which three or more filter assemblies 200 are connected in parallel.

In this case, the added filter assembly 200 includes the culture medium supply line 102, the vent line 106, the gas supply line 104 connected to the gas tank and the harvest line 114, respectively, and the extrusion line 112, as described above. ) is installed.

In order to manufacture cell-derived endoplasmic reticulum using the cell-derived endoplasmic reticulum manufacturing apparatus 40 according to the fourth embodiment of the present invention, first, the first filter assembly 200 and the second filter assembly 200' through the culture medium supply line The cell culture medium is introduced into one or more of the filter assemblies. Thereafter, the gas supply lines 104 and 104' connected to the gas tank are opened and the vent lines 106 and 106' are closed so that the cell culture medium passes through the cartridge filter 220 inside the filter assembly 200 and 200' under a predetermined pressure, Through the harvest line 114, spent media without remaining cells is discharged. Next, in a state where the gas supply lines 104 and 104' are closed, a solution suitable for suspending cells such as phosphate buffered saline (PBS) is injected through the harvest line 114 to filter assembly ( 200,200') to form a cell suspension.

Subsequently, the gas supply line 104 provided in the first filter assembly 200 is opened and the vent line 106 is closed. In addition, the gas supply line 104' provided in the second filter assembly 200' is closed and the vent line 106' is opened so that the cell suspension is supplied to the first filter assembly 200 and the second filter assembly 200'. ) to pass through the cartridge filter 220 inside.

Then, the gas supply line 104 connected to the first filter assembly 200 is closed and the vent line 106 is opened. In addition, the gas supply line 104' provided in the second filter assembly 200' is opened and the vent line 106' is closed so that the cell suspension accommodated in the second filter assembly 200' is removed from the previous step. It flows in the opposite direction, that is, in the direction from the second filter assembly 200' to the first filter assembly 200, and passes through the cartridge filter 220.

As such, the process of alternately opening and closing the gas supply lines 104 and 104' is repeated a predetermined number of times to allow the cell suspension to reciprocate through the filter 220, thereby increasing the production efficiency of cell-derived endoplasmic reticulum.

After allowing the cell suspension to pass through the filter 220 repeatedly a predetermined number of times, the harvest line 114 is opened to obtain a final extrusion solution.

In this case, the final extrusion solution may include cell-derived endoplasmic reticulum.

As described above, in the process of manufacturing cell-derived endoplasmic reticulum using the cell-derived endoplasmic reticulum manufacturing apparatus 40 according to the fourth embodiment of the present invention, since cell harvesting and extrusion are performed only within the cell-derived endoplasmic reticulum manufacturing apparatus, the cells are externally The risk of contamination or deformation due to exposure to the environment is reduced, the process can be simplified, and the process speed is improved.

Meanwhile, in the cell-derived endoplasmic reticulum manufacturing method, instead of the cell culture medium, the cell suspension may be introduced into the filter assemblies 200 and 200' through the culture medium supply lines 102 and 102'. At this time, the spent media in which cells do not remain are discharged through the harvest line 114 and a solution suitable for resuspending the cells is injected through the harvest line 114 into the filter assembly 200 or 200'. The step of forming a cell suspension may be omitted. In addition, at this time, the cell suspension may be a solution obtained by harvesting cells from a cell culture medium through centrifugation and resuspending cells in a solution suitable for resuspending cells such as PBS.

Hereinafter, examples of experiments performed using the cell-derived endoplasmic reticulum manufacturing apparatuses 10, 20, 30, and 40 according to the first to fourth embodiments of the present invention will be described.

In the experimental example, particle concentration and size measurement, PDI measurement, and protein and DNA concentration measurement were commonly performed as follows.

Particle concentration and size measurement

The concentration and size of the particles were measured using Zetaview equipment (Particle metric). Equipment preparation and cleaning procedures were carried out according to the manufacturer's instructions. Samples prepared for analysis were diluted using a PBS solution passed through a 0.1 μm pore filter to 50 to 200 particles/frame, and particle concentration and particle size were measured using 1 mL of the sample.

PDI measurement

PDI was measured using a Zatasizer NanoZS (Malvern). The prepared sample was diluted using a PBS solution passed through a 0.1 μm pore filter to obtain a 600-1200 derived count rate, and then 1-2 mL of the sample was placed in a cuvette (Kartell) and measured.

Measurement of protein concentration and DNA concentration

Protein and DNA concentrations were measured using a Qubit™ 4 Fluorometer (Invitrogen). For the prepared samples, Qubit™ Protein Assay Kit and Qubit™ dsDNA HS Assay Kit were used to measure protein and DNA concentrations, respectively. The sample and standard solutions were mixed with the working solution by vortexing, and the solution for protein measurement was reacted for 15 minutes and the solution for DNA measurement for 2 minutes at room temperature. Afterwards, each reacted solution was placed in a Qubit™ 4 Fluorometer chamber and the final concentration was measured according to the manufacturer's instructions.

Using the cell-derived endoplasmic reticulum manufacturing apparatus 10 according to the first embodiment of the present invention, a test for cell harvesting was performed as in <Experimental Example 1> below.

Experimental Example 1

For cell harvest, one depth filter (Sartopure PP3 Capsules, total membrane area: 0.026 m ² , retention rates: 0.65 ㎛) is connected to a pressure vessel, and air is supplied to the pressure vessel. Connect the gas supply line. 500 mL of HEK293 cell culture medium concentration 5.78E+06 cells/mL was put into a pressure vessel. The medium (spent media) used in the harvest line was discharged by pressurizing with 0.1 bar of air. The draining time of the medium used was 120 seconds.

For the analysis of cell retention in the used medium, 5 to 10 mL was sampled after sufficiently mixing the medium used physically, and the sample was evenly distributed on the surface of a 100mm culture petri-dish for cell observation. Then, visual inspection was performed at 4X, 10X, and 40X magnifications using an Olympus CKX53 inverted microscope. As a result of the confirmation, it was analyzed that there were no remaining cells in the medium used.

Using the cell-derived endoplasmic reticulum manufacturing apparatus 20 according to the second embodiment of the present invention, the cell extrusion process was performed as shown in <Experimental Examples 2 to 17> below.

Experimental Example 2

For cell extrusion, two depth filters (Sartopure PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) were connected between pressure vessels, and a gas supply line was connected to each pressure vessel to supply air. connected. HEK293 cell culture medium concentration 7.87E+06 cells/mL 500 mL was harvested by centrifugation. The harvested cells were resuspended in 500 mL room temperature phosphate buffered saline (PBS), and then put into two pressure vessels in half. One valve of the gas supply line was closed and the other was opened so that the cell solution resuspended in one direction passed through the filter. Opening and closing the left and right valves in opposite directions was repeated a total of 60 times. The air pressure supplied to the pressure vessel was 1 bar for the first extrusion and 2.5 bar for 2 to 60 extrusions. The particle concentration in the final extrusion solution was measured, and the productivity of particles per cell was analyzed as 11,563 particles/cell.

Experimental Example 3

For cell extrusion, two depth filters (Sartopure PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) are connected between pressure vessels, and a gas supply line is connected to each pressure vessel to supply air. did 500 mL of HEK293 cell culture medium concentration 7.65E+06 cells/mL was harvested by centrifugation. The harvested cells were resuspended in 500 mL of room temperature PBS, and then put into two pressure vessels in half. One valve of the gas supply line was closed and the other was opened so that the cell solution resuspended in one direction passed through the filter. The left and right valves were opened and closed in reverse, and a total of 100 times were passed. The air pressure supplied to the pressure vessel was 1 bar for the first extrusion and 2.5 bar for 2 to 100 extrusions. The particle concentration in the final extrusion solution was measured, and the productivity of particles per cell was analyzed as 17,300 particles/cell.

Experimental Example 4

For cell extrusion, two depth filters (Sartopure PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) are connected between pressure vessels, and a gas supply line is connected to each pressure vessel to supply air. did 300 mL of HEK293 cell culture medium concentration 5.80E+06 cells/mL was harvested by centrifugation. The harvested cells were resuspended in 500 mL of room temperature PBS, and then put into two pressure vessels in half. One valve of the gas supply line was closed and the other was opened so that the cell solution resuspended in one direction passed through the filter. Opening and closing the left and right valves in opposite directions was repeated a total of 90 times. The air pressure supplied to the pressure vessel was 1 bar for the first extrusion and 2.5 bar for extrusions 2 to 90 times. The particle concentration in the final extrusion solution was measured, and the productivity of particles per cell was analyzed as 17,241 particles/cell.

Experimental Example 5

For cell extrusion, two depth filters (Sartopure PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) are connected between pressure vessels, and a gas supply line is connected to each pressure vessel to supply air. did HEK293 cell culture medium concentration 3.15E+06 cells/mL 584 mL was put into two pressure vessels in half. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge spent media to the harvest line. The medium drain time used was 29 seconds. For the analysis of cell retention in the used medium, 5 to 10 mL was sampled after sufficiently mixing the medium used physically, and the sample was evenly distributed on the surface of a 100 mm culture petri-dish for cell observation. Then, it was visually confirmed at 4X, 10X, and 40X magnification using an Olympus CKX53 inverted microscope, and it was analyzed that there were no cells remaining in the medium used. Back-flushing, which is a process of resuspending the harvested cells inside the extrusion device, was performed by injecting 300 mL of PBS at room temperature back into the harvest line using a peristaltic pump. Back-flushing time took a total of 81 seconds. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. Opening and closing the left and right valves in opposite directions was repeated a total of 120 times. The air pressure supplied to the pressure vessel was carried out at 1.0 bar. The particle concentration in the final extrusion solution was measured and the productivity of particles per cell was analyzed as 15.492 particles/cell.

Experimental Example 6

For cell extrusion, two depth filters (Sartopure PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) are connected between pressure vessels, and a gas supply line is connected to each pressure vessel to supply air. did 500 mL of HEK293 cell culture medium concentration 3.15E+06 cells/mL was put into two pressure vessels in half. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge spent media to the harvest line. The medium drain time used was 30 seconds. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. 300 mL of room temperature PBS was back-flushed into the harvest line using a peristaltic pump. The back-flushing time took a total of 90 seconds. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. The left and right valves were opened and closed in reverse, and a total of 100 times were passed. The air pressure supplied to the pressure vessel was 1.0 bar for the first and 2.5 bar for the 2nd to 100th. Particle concentration, particle size, PDI, protein concentration, and DNA concentration in the final extrusion solution were measured, and the productivity of particles per cell was analyzed as 30,476 particles/cell. The analysis results for this are as shown in FIG. 7 .

Experimental Example 7

For cell extrusion, two depth filters (Sartopure PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) are connected between pressure vessels, and a gas supply line is connected to each pressure vessel to supply air. did 400 mL of HEK293 cell culture medium concentration 7.62E+06 cells/mL was put into two pressure vessels in half each. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge the used medium to the harvest line. The medium drain time used was 33 seconds. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. 400 mL of room temperature PBS was back-flushed to the harvest line using a peristaltic pump. The back-flushing time took a total of 120 seconds. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. Opening and closing the left and right valves in opposite directions was repeated a total of 80 times. The air pressure supplied to the pressure vessel was 1.0 bar for the first and 2.5 bar for the 2nd to 80th. The particle concentration in the final extrusion solution was measured and the productivity of particles per cell was analyzed as 23,622 particles/cell.

Experimental Example 8

For cell extrusion, two depth filters (Sartopure PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) are connected between pressure vessels, and a gas supply line is connected to each pressure vessel to supply air. did 390 mL of HEK293 cell culture medium concentration 7.52E+06 cells/mL was put in half each into two pressure vessels. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge the used medium to the harvest line. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. 200 mL of room temperature PBS was back-flushed to the harvest line using a peristaltic pump. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. Opening and closing the left and right valves in opposite directions was repeated a total of 80 times. The air pressure supplied to the pressure vessel was 1.0 bar for the first and 2.5 bar for the 2nd to 80th. The particle concentration in the final extrusion solution was measured and the productivity of particles per cell was analyzed as 12,275 particles/cell.

Experimental Example 9

For cell extrusion, two depth filters (Sartopure PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) were connected between the pressure vessels, and a gas supply line was used to supply nitrogen (N ₂ ) gas to each pressure vessel. connected. 867 mL of HEK293 cell culture medium concentration 3.46E+06 cells/mL was put into two pressure vessels in half each. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge the used medium to the harvest line. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. 400 mL of room temperature PBS was back-flushed to the harvest line using a peristaltic pump. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. Opening and closing the left and right valves in opposite directions was repeated a total of 70 times. The nitrogen gas pressure supplied to the pressure vessel was 1.0 bar for the first time and 2.5 bar for the 2nd to 70th times. The average extrusion time was measured as 36 seconds. The particle concentration in the final extrusion solution was measured and the productivity of particles per cell was analyzed as 24,001 particles/cell.

Experimental Example 10

For cell extrusion, two depth filters (Sartopure PP3 MiDicaps, total membrane area: 0.1 m ² , retention rates: 0.65 ㎛) were connected between the pressure vessels, and a gas supply line was used to supply nitrogen (N ₂ ) gas to each pressure vessel. connected. HEK293 cell culture medium concentration 8.13E+06 cells/mL 643 mL was put into two pressure vessels in half. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge the used medium to the harvest line. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. 800 mL of room temperature PBS was back-flushed to the harvest line using a peristaltic pump. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. The left and right valves were opened and closed in reverse, and a total of 50 times were passed. The nitrogen gas pressure supplied to the pressure vessel was 1.0 bar for the first time and 2.5 bar for the 2nd to 50th times. The average extrusion time was measured as 56 seconds. The particle concentration in the final extrusion solution was measured and the productivity of particles per cell was analyzed as 22,955 particles/cell.

Experimental Example 11

For cell extrusion, two depth filters (Sartopure PP3 MiDicaps, total membrane area: 0.1 m ² , retention rates: 0.65 ㎛) were connected between pressure vessels, and a gas supply line for supplying nitrogen (N ₂ ) gas to each pressure vessel. connected. HEK293 cell culture medium concentration 8.09E+06 cells/mL 793 mL was put into two pressure vessels in half. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge the used medium to the harvest line. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. 800 mL of room temperature PBS was back-flushed to the harvest line using a peristaltic pump. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. Opening and closing the left and right valves in opposite directions was repeated a total of 70 times. The nitrogen gas pressure supplied to the pressure vessel was 1.0 bar for the first time and 2.5 bar for the 2nd to 70th times. The average extrusion time was measured as 47 seconds. The particle concentration in the final extrusion solution was measured and the productivity of particles per cell was analyzed as 34,916 particles/cell.

Experimental Example 12

For cell extrusion, two depth filters (Sartopure PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) were connected between pressure vessels, and a gas supply line for supplying nitrogen (N ₂ ) gas to each pressure vessel. connected. 575 mL of HEK293 cell culture medium concentration 5.22E+06 cells/mL was put into two pressure vessels in half each. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge the used medium to the harvest line. The media evacuation time used was 4 minutes. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. 800 mL of room temperature PBS was back-flushed to the harvest line using a peristaltic pump. The back-flushing time took 2 minutes in total. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. Opening and closing the left and right valves in opposite directions was repeated a total of 80 times. The nitrogen gas pressure supplied to the pressure vessel was 1.0 bar for the first time and 2.5 bar for the 2nd to 80th times. The particle concentration in the final extrusion solution was measured and the productivity of particles per cell was analyzed as 18,124 particles/cell.

Experimental Example 13

For cell extrusion, two depth filters (Sartopure PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) were connected between pressure vessels, and a gas supply line for supplying nitrogen (N ₂ ) gas to each pressure vessel. connected. 575 mL of HEK293 cell culture medium concentration 5.22E+06 cells/mL was put into two pressure vessels in half each. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge the used medium to the harvest line. The media evacuation time used was 4 minutes. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. Using a peristaltic pump, 1,600 mL of room temperature PBS was back-flushed into the harvest line. The back-flushing time took a total of 4 minutes. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. Opening and closing the left and right valves in opposite directions was repeated a total of 70 times. The nitrogen gas pressure supplied to the pressure vessel was 1.0 bar for the first time and 2.5 bar for the 2nd to 70th times. The particle concentration in the final extrusion solution was measured and the productivity of particles per cell was analyzed as 23,389 particles/cell.

Experimental Example 14

For cell extrusion, two depth filters (Sartopure PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) were connected between pressure vessels, and a gas supply line for supplying nitrogen (N ₂ ) gas to each pressure vessel. connected. 575 mL of HEK293 cell culture medium concentration 5.22E+06 cells/mL was put into two pressure vessels in half each. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge the used medium to the harvest line. The media evacuation time used was 4 minutes. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. Using a peristaltic pump, 800 mL of phosphate-buffered saline water at 37 ° C was back-flushed to the harvest line. The back-flushing time took 2 minutes in total. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. Opening and closing the left and right valves in opposite directions was repeated a total of 80 times. The nitrogen gas pressure supplied to the pressure vessel was 1.0 bar for the first time and 2.5 bar for the 2nd to 80th times. The particle concentration in the final extrusion solution was measured and the productivity of particles per cell was analyzed as 23,988 particles/cell.

Experimental Example 15

For cell extrusion, two depth filters (Sartopure® PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) are connected between pressure vessels, and nitrogen (N ₂ ) gas is supplied to each pressure vessel. line was connected. HEK293 cell culture medium concentration 4.56E+06 cells/mL 658 mL was put into two pressure vessels, half each. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge the used medium to the harvest line. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. Using a peristaltic pump, 1,600 mL of 37° C. PBS was back-flushed into the harvest line. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. Opening and closing the left and right valves in opposite directions was repeated a total of 70 times. The nitrogen gas pressure supplied to the pressure vessel was 1.0 bar for the first time and 2.5 bar for the 2nd to 70th times. The average extrusion time was measured as 68 seconds. The particle concentration in the final extrusion solution was measured and the productivity of particles per cell was analyzed as 26,146 particles/cell.

Experimental Example 16

For cell extrusion, two depth filters (Sartopure PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) were connected between pressure vessels, and a gas supply line for supplying nitrogen (N ₂ ) gas to each pressure vessel. connected. 684 mL of HEK293 cell culture medium concentration 4.38E+06 cells/mL was put into two pressure vessels in half. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge the used medium to the harvest line. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. Using a peristaltic pump, 800 mL of 37 °C PBS was back-flushed into the harvest line. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. Opening and closing the left and right valves in opposite directions was repeated a total of 70 times. The nitrogen gas pressure supplied to the pressure vessel was 1.0 bar for the first time and 2.5 bar for the 2nd to 70th times. The average extrusion time was measured as 37 seconds. The particle concentration in the final extrusion solution was measured and the productivity of particles per cell was analyzed as 23,800 particles/cell.

Experimental Example 17

For cell extrusion, two depth filters (Sartopure PP3 Capsules, total membrane area: 0.052 m ² , retention rates: 0.65 ㎛) were connected between pressure vessels, and a gas supply line for supplying nitrogen (N ₂ ) gas to each pressure vessel. connected. 667 mL of HEK293 cell culture medium concentration 4.80E+06 cells/mL was put into two pressure vessels in half. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge the used medium to the harvest line. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. Using a peristaltic pump, 400 mL of 37° C. PBS was back-flushed into the harvest line. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. Opening and closing the left and right valves in opposite directions was repeated a total of 70 times. The nitrogen gas pressure supplied to the pressure vessel was 1.0 bar for the first time and 2.5 bar for the 2nd to 70th times. The average extrusion time was measured as 21 seconds. The particle concentration in the final extrusion solution was measured and the productivity of particles per cell was analyzed as 16,374 particles/cell.

Using the cell-derived endoplasmic reticulum manufacturing apparatus 30 according to the third embodiment of the present invention, the cell extrusion process was performed as in <Experimental Example 18> below.

Experimental Example 18

For cell extrusion, one depth filter (Sartopure PP3 MiDicaps, total membrane area: 0.05 m ² , retention rates: 0.65 ㎛) was connected between pressure vessels, and a gas supply line for supplying nitrogen (N ₂ ) gas to each pressure vessel. connected. 620 mL of HEK293 cell culture medium concentration 3.72E+06 cells/mL was put into a pressure vessel connected to the forward direction of the depth filter. Both valves of the gas supply line were opened and pressurized at 0.2 bar to discharge the used medium to the harvest line. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. Using a peristaltic pump, 615 mL of 37° C. PBS was back-flushed into the harvest line. For extrusion, one valve of the gas supply line was closed and the other valve was opened to allow the resuspended cell solution to pass through the filter in one direction. Opening and closing the left and right valves in opposite directions was repeated a total of 70 times. The nitrogen gas pressure supplied to the pressure vessel was 1.0 bar for the first time and 2.5 bar for the 2nd to 70th times. The average extrusion time was measured as 37 seconds. The particle concentration in the final extrusion solution was measured and the productivity of particles per cell was analyzed as 29,343 particles/cell.

Using the cell-derived endoplasmic reticulum manufacturing apparatus 40 according to the fourth embodiment of the present invention, the cell extrusion process was performed as shown in <Experimental Examples 19 to 22> below, [Table 1] shows <Experimental Examples 20 to 22> It shows the results of crude-CDV mass production according to

division dP
(bar) 1 time average extrusion
process time
(candle) Recovery weight (kg) Particle concentration (×10 ¹¹ particles/mL) Size (nm) PDI DNA
(μg/mL) productivity
Y _P/C Experimental Example 20 0.20 36.8 17.3 0.84 159.1 0.285 4.80 17,920 Experimental Example 21 0.26 28.3 18.4 0.63 159.3 0.314 5.02 13,440 Experimental Example 22 0.19 30.9 18.7 0.69 164.6 0.333 4.80 14,720 Mean 0.22 32.0 18.1 0.72 161 0.311 4.87 15,360 STDEV 0.04 4.4 0.7 0.11 3 0.024 0.13 2,308 % CV 17.5 13.6 4.1 15.0 1.9 7.8 2.6 15.0 dP = average of the difference between the maximum and minimum pressures measured in the extrusion solution during extrusion

Experimental Example 19

For cell extrusion, a total of four cartridge depth filters (Sartopure PP3 Cartridge, total membrane area: 1.8 m ² , retention rates: 0.65 μm) were mounted on the filter assembly. Specifically, two cartridge depth filters were mounted on each lower end of the two filter assemblies, and the idle mounting hole was closed with a cap. Thereafter, a gas supply line for supplying nitrogen (N ₂ ) gas was connected to each filter assembly. HEK293 cell culture medium concentration 6.00E+06 cells/mL 12.5 L was put into two filter assemblies, half each. Both valves of the gas supply line were opened and pressurized at 0.4 bar to discharge the used medium to the harvest line. The media evacuation time used was 5 minutes. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. 20 L of 37° C. PBS was back-flushed into the harvest line using a peristaltic pump. The back-flushing time took a total of 16 minutes. For extrusion, one valve of the gas supply line was closed and the other valve was opened so that the resuspended cell solution in one direction passed through the cartridge filter inside the filter assembly. Opening and closing the left and right valves in opposite directions was repeated a total of 70 times. The nitrogen gas pressure supplied to the filter assembly was 1.0 bar for the first and 2.5 bar for the 2nd to 70th. The average extrusion time was measured as 32.2 seconds. Particle concentration, particle size, PDI, and DNA concentration in the final extrusion solution were measured, and the productivity of particles per cell was analyzed as 17,493 particles/cell.

Experimental Example 20

For cell extrusion, a total of four cartridge depth filters (Sartopure PP3 Cartridge, total membrane area: 1.8 m ² , retention rates: 0.65 μm) were mounted on the filter assembly. Specifically, two cartridge depth filters were mounted on each lower end of the two filter assemblies, and the idle mounting hole was closed with a cap. Thereafter, a gas supply line for supplying nitrogen (N ₂ ) gas was connected to each filter assembly. HEK293 cell culture medium concentration 6.00E+06 cells/mL 12.5 L was put into two filter assemblies, half each. Both valves of the gas supply line were opened and pressurized at 0.4 bar to discharge the used medium to the harvest line. The medium drain time used was 22 minutes. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. 16 L of 37° C. PBS was back-flushed into the harvest line using a peristaltic pump. The back-flushing time took a total of 9 minutes. For extrusion, one valve of the gas supply line was closed and the other valve was opened so that the resuspended cell solution in one direction passed through the cartridge filter inside the filter assembly. Opening and closing the left and right valves in opposite directions was repeated a total of 70 times. The nitrogen gas pressure supplied to the filter assembly was 1.0 bar for the first and 2.5 bar for the 2nd to 70th. The average extrusion time was measured as 36.8 seconds. Particle concentration and particle size in the final extrusion solution were measured, and the productivity of particles per cell was analyzed as 17,920 particles/cell.

Experimental Example 21

For cell extrusion, a total of four cartridge depth filters (Sartopure PP3 Cartridge, total membrane area: 1.8 m ² , retention rates: 0.65 μm) were mounted on the filter assembly. Specifically, two cartridge depth filters were mounted on each lower end of the two filter assemblies, and the idle mounting hole was closed with a cap. Thereafter, a gas supply line for supplying nitrogen (N ₂ ) gas was connected to each filter assembly. HEK293 cell culture medium concentration 6.00E+06 cells/mL 12.5 L was put into two filter assemblies, half each. Both valves of the gas supply line were opened and pressurized at 0.4 bar to discharge the used medium to the harvest line. The media evacuation time used was 19 minutes. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. 16 L of 37° C. PBS was back-flushed into the harvest line using a peristaltic pump. The back-flushing time took a total of 9 minutes. For extrusion, one valve of the gas supply line was closed and the other valve was opened so that the resuspended cell solution in one direction passed through the cartridge filter inside the filter assembly. Opening and closing the left and right valves in opposite directions was repeated a total of 70 times. The nitrogen gas pressure supplied to the filter assembly was 1.0 bar for the first and 2.5 bar for the 2nd to 70th. The average extrusion time was measured as 28.3 seconds. Particle concentration, particle size, PDI, and DNA concentration in the final extrusion solution were measured, and the productivity of particles per cell was analyzed as 13,440 particles/cell.

Experimental Example 22

For cell extrusion, a total of four cartridge depth filters (Sartopure PP3 Cartridge, total membrane area: 1.8 m ² , retention rates: 0.65 μm) were mounted on the filter assembly. Specifically, two cartridge depth filters were mounted on each lower end of the two filter assemblies, and the idle mounting holes were closed with caps. Thereafter, a gas supply line for supplying nitrogen (N ₂ ) gas was connected to each filter assembly. HEK293 cell culture medium concentration 6.00E+06 cells/mL 12.5 L was put into two filter assemblies, half each. Both valves of the gas supply line were opened and pressurized at 0.4 bar to discharge the used medium to the harvest line. The media evacuation time used was 21 minutes. As a result of analyzing the medium used by the method for analyzing cell retention in the used medium described in Experimental Example 5, it was analyzed that there were no cells remaining in the used medium. 16 L of 37° C. PBS was back-flushed into the harvest line using a peristaltic pump. The back-flushing time took a total of 9 minutes. For extrusion, one valve of the gas supply line was closed and the other valve was opened so that the resuspended cell solution in one direction passed through the cartridge filter inside the filter assembly. Opening and closing the left and right valves in opposite directions was repeated a total of 70 times. The nitrogen gas pressure supplied to the filter assembly was 1.0 bar for the first and 2.5 bar for the 2nd to 70th. The average extrusion time was measured as 30.9 seconds. Particle concentration, particle size, PDI, and DNA concentration in the final extrusion solution were measured, and the productivity of particles per cell was analyzed as 14,720 particles/cell.

Meanwhile, FIG. 8 is a diagram of the productivity of particles per cell according to various parameters, FIG. 9 is a diagram of the average extrusion processing time result according to various parameters, and FIG. 10 is a diagram of the amount of PBS per cell and the temperature of PBS. This is a diagram of the results of the productivity of particles per cell and the average extrusion process time per cell according to the results. As shown, it can be confirmed that mass production of cell-derived endoplasmic reticulum is possible with high efficiency using the apparatus for manufacturing cell-derived endoplasmic reticulum of the present invention.

As described above, the cell-derived endoplasmic reticulum manufacturing apparatus and manufacturing method using the same of the present invention can reduce the risk of contamination and deformation by preparing cell-derived endoplasmic reticulum in an environment where external exposure is minimized, and can be stably, economically, and mass-produced. It is very beneficial because the production capacity can be easily adjusted according to this.

The above description is merely an example of the technical idea of the present invention, and those skilled in the art can make various modifications, changes, and substitutions without departing from the essential characteristics of the present invention. will be. Therefore, the embodiments disclosed in the present invention and the accompanying drawings are not intended to limit the technical idea of the present invention, but to explain, and the scope of the technical idea of the present invention is not limited by these embodiments and the accompanying drawings. The protection scope of the present invention should be construed by the claims, and all technical ideas within the equivalent range should be interpreted as being included in the scope of the present invention.

10,20,30,40: cell-derived endoplasmic reticulum production device
100: pressure vessel 102: culture solution supply line
104: gas supply line 106: vent line
110: filter 112: extrusion line
114: harvest line 120: valve
200: filter assembly
210: lower part 212: mounting hole
214: cap 220: cartridge filter
230: middle part 240: upper part

Claims (20)

a) extruding the cell culture medium through a depth filter; and
b) obtaining cell-derived endoplasmic reticulum from the extruded solution; including, cell-derived endoplasmic reticulum production method using a depth filter.
According to claim 1,
Characterized in that the extruding step of a) is repeated 1 to 100 times, a cell-derived endoplasmic reticulum manufacturing method using a depth filter.
According to claim 2,
Characterized in that the extruding step is performed while changing the pressure, cell-derived endoplasmic reticulum manufacturing method using a depth filter.
According to claim 3,
Characterized in that the extrusion is performed by increasing the pressure of the extrusion step after the second round than the pressure of the first extrusion step, cell-derived endoplasmic reticulum manufacturing method using a depth filter.
a) extruding the cell culture medium through a depth filter under a first pressure; and
b) extruding the solution extruded in step a) by passing it through a depth filter under a second pressure;
According to claim 5,
In the step b), the cell-derived endoplasmic reticulum manufacturing method characterized in that the extruded solution is passed through the depth filter back and forth a plurality of times.
According to claim 5,
The second pressure is a cell-derived endoplasmic reticulum manufacturing method, characterized in that higher than the first pressure.
According to claim 7,
The first pressure is 1 bar, the second pressure is a method for producing cell-derived endoplasmic reticulum, characterized in that 2.5 bar.
According to claim 5,
The extruded solution in step b) is a solution resuspended in a cell suspension after being extruded in step a), a method for producing cell-derived vesicles.
According to claim 9,
The PBS is 20 to 37 ℃, cell-derived endoplasmic reticulum production method.
According to claim 5,
The total film area of the depth filter is 0.026 to 0.1 m ² ,
The first pressure is 0.2 bar, the cell-derived endoplasmic reticulum manufacturing method, characterized in that the second pressure is 2.5 bar.
According to claim 5,
The total membrane area of the depth filter is 0.45 to 2.7 m ² ,
The first pressure is 0.4 bar, the cell-derived endoplasmic reticulum manufacturing method, characterized in that the second pressure is 2.5 bar.
According to claim 5,
The second pressure is
The cell-derived endoplasmic reticulum manufacturing method, characterized in that 1 bar during the first cell extrusion in step b) and 2.5 bar during subsequent cell extrusion.
According to claim 9,
The method for producing cell-derived vesicles, characterized in that the cell suspension is injected in a direction opposite to the direction in which the cell culture solution is discharged from the depth filter.
According to any one of claims 1 to 14,
The cell-derived endoplasmic reticulum manufacturing method is characterized in that cell extrusion and cell-derived endoplasmic reticulum harvesting are performed in one cell-derived endoplasmic reticulum manufacturing apparatus, cell-derived endoplasmic reticulum manufacturing method.
The method of claim 15, wherein the cell-derived endoplasmic reticulum manufacturing apparatus
a first pressure vessel and a second pressure vessel to which a culture medium supply line and a gas supply line are connected to one side of which a cell culture medium is supplied;
a first extrusion line connected to the other side of the first pressure vessel;
a second extrusion line connected to the other side of the second pressure vessel and communicating with the first extrusion line;
at least one depth filter disposed on the first extrusion line or the second extrusion line through which the cell culture medium passes; and
A method for producing cell-derived vesicles comprising a harvest line communicating with the first extrusion line and the second extrusion line.
According to claim 16,
The method for producing cell-derived vesicles, characterized in that at least one depth filter is disposed on the first extrusion line and the second extrusion line.
The method of claim 15, wherein the cell-derived endoplasmic reticulum manufacturing apparatus
A first filter assembly and a second filter assembly having a culture medium supply line and a gas supply line connected to one side of which a cell culture medium is supplied and having a depth filter therein;
a first extrusion line connected to the other side of the first filter assembly;
a second extrusion line connected to the other side of the second filter assembly and communicating with the first extrusion line; and
A method for producing cell-derived vesicles comprising a harvest line communicating with the first extrusion line and the second extrusion line.
According to claim 18,
The first filter assembly and the second filter assembly
a lower portion in which at least one mounting hole in which the depth filter is mounted is formed;
a middle portion formed to accommodate the depth filter therein and coupled to the lower portion; and
A method for producing cell-derived endoplasmic reticulum, comprising an upper end fastened to an upper end of the middle part to seal the inside together with the lower end and the middle part.
According to claim 19,
The first filter assembly and the second filter assembly
The method of manufacturing cell-derived endoplasmic reticulum, characterized in that further comprising a cap detachably provided to the mounting hole and sealing the mounting hole in a state inserted into the mounting hole.

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