KR20190124608A - Automated cell culturing method - Google Patents

Abstract

The present invention relates to an automatic cell cultivation method to cultivate cells in a system, which comprises a storage unit, a main processing unit, a constant-temperature cultivation unit, and a control unit, by the control of the control unit. According to the present invention, the method comprises: a step of thawing a vial having a cell of a frozen state contained therein; a step of charging the cells into a first cultivation vessel to cultivate the cell in the constant temperature cultivation unit; a step of moving the first cultivation vessel cultivated in the constant temperature cultivation unit to the main processing unit and then, exchanging a cultivation solution; a step of performing cultivation in the constant temperature cultivation unit; a step of separating the cells from a cultivation surface of the first cultivation vessel and charging the cells into a second cultivation vessel; a step of cultivating the cells in the constant temperature cultivation unit; and a step of separating the cells from a cultivation surface of the second cultivation vessel and capturing the cells.

Description

Automated cell culture method {AUTOMATED CELL CULTURING METHOD}

The present invention relates to a method for automating cell culture, and more particularly, to a method for automating cell culture to obtain a larger amount of cells by automating the production of cells used in the production of cell therapeutics.

Generally, cell therapies include a series of autologous, allogenic, and xenogenic cells that can be expanded or screened in vitro to alter the function of cells and tissues, or by altering the biological characteristics of the cells. Medicines used for the purpose of treatment, diagnosis and prevention through the act of.

Conventionally, such cell therapy products are produced manually, but the number of cell therapy products obtained by manual production is limited and there are problems such as differences in culture performance and quality of cells depending on the worker. A device that mass-produces uniform, high-quality cells by implementing optimized culture conditions in an automated process plays an important role in the industrialization and standardization of cell therapy production.

The technical problem to be solved by the present invention is to provide a cell culture method capable of constantly culturing a large amount of cells through an automated process.

In order to solve the technical problem as described above, in a system comprising a storage unit, a main working unit, a constant temperature culture unit and a control unit, the method of an embodiment of the present invention for culturing cells under the control of the control unit, ( a) thawing the vial containing the frozen cells, aliquoting the first culture vessel and culturing in the incubation; (b) moving the first culture vessel cultured in the incubation portion to the main working portion, exchanging a culture solution, and culturing in the incubation portion; (c) separating the cells from the culture surface of the first culture vessel, aliquoting the second culture vessel, and culturing in the incubation portion; And (d) separating the cells from the culture surface of the second culture vessel and collecting the cells.

In one embodiment of the present invention, the step (a) comprises the steps of: (a-1) placing the vial containing the frozen cells in the heating unit of the main work unit; (a-2) adding washing solution to the first tube through tubing, and adding the thawed cells from the vial to the first tube with a tip; (a-3) placing the first tube in a centrifuge, driving the centrifuge to spin down, and removing the supernatant using a tip; (a-4) adding a culture solution to the first tube through a tip to prepare a cell solution by mixing the cell pellet and the culture solution; (a-5) dispensing the culture solution through the tubing in the first culture vessel; (a-6) dispensing the cell solution into the first culture vessel; (a-7) driving the culture vessel handler to arrange and handle the culture surface of the first culture vessel in a state perpendicular to the bottom surface of the main working portion; (a-8) driving the culture vessel handler to arrange and handle the culture surface of the first culture vessel in parallel with the bottom surface of the main working portion; And (a-9) moving the first culture vessel to the incubation portion.

In one embodiment of the present invention, the culture solution is stored in a predetermined container in the storage unit, it may be provided to the first tube or the first culture vessel of the main working portion through a tube from the predetermined container.

In one embodiment of the present invention, the step (b) comprises the steps of (b-1) driving the culture vessel handler to remove the culture solution inside the first culture vessel; (b-2) dispensing a new culture solution through tubing to the first culture vessel; And (b-3) driving the culture vessel handler to arrange and handle the culture surface of the first culture vessel in parallel with the bottom surface of the main work portion.

In one embodiment of the present invention, (b-4) may further comprise the step of receiving the image of the culture surface of the first culture vessel from the microscope to determine the density and shape of the cells of the cells.

In one embodiment of the present invention, the density and shape of the cells may be determined by comparing the image received from the microscope with the reference image, or by calculating the ratio of the area occupied by the cells.

In one embodiment of the present invention, the step (c), (c-1) driving the culture vessel handler to remove the culture medium in the first culture vessel; (c-2) adding the washing liquid to the first culture vessel through tubing; (c-3) driving the culture vessel handler to arrange and handle the culture surface of the first culture vessel parallel to the bottom surface of the main working portion; (c-4) driving the culture vessel handler to remove the washing solution in the first culture vessel; (c-5) adding a cell separator to the first culture vessel, and driving the culture vessel handler to arrange and handle the culture surface of the first culture vessel parallel to the bottom surface of the main working portion; (c-6) placing the first culture vessel in the constant temperature culture portion for a predetermined time; (c-7) driving the culture vessel handler to add the culture solution to the first culture vessel and to arrange and handle the culture surface of the first culture vessel parallel to the bottom surface of the main work unit; (c-8) dispensing the cells remaining in the first culture vessel in a second tube; (c-9) arranging the second tube in a centrifuge, driving the centrifuge to spin down, removing the supernatant using a tip, and floating the remaining cell pellet in a culture medium to prepare a cell suspension; (c-10) adding a culture solution to the mixing vessel through tubing, and adding the cell suspension of the first culture vessel to the mixing vessel using a tip to prepare a suspension solution; (c-11) driving the culture vessel handler to arrange and handle the culture surface of the mixing vessel perpendicular to the bottom; And (c-12) dispensing the suspension solution of the mixed vessel into a second culture vessel and moving to the constant temperature culture portion.

In one embodiment of the present invention, the step (c), (c-13) after a predetermined time elapses after the step (c-5), the culture surface of the first culture vessel is the bottom surface of the main working part In a state arranged in parallel with the, may further comprise the step of applying a physical impact on the left and right of the culture surface.

In one embodiment of the present invention, the step (d) comprises the steps of (d-1) driving the culture vessel handler to remove the culture medium inside the second culture vessel; (d-2) adding the washing liquid to the second culture vessel through tubing; (d-3) driving the culture vessel handler to arrange and handle the culture surface of the second culture vessel parallel to the bottom surface of the main working portion; (d-4) driving the culture vessel handler to remove the washing solution in the second culture vessel; (d-5) driving the culture vessel handler to add the cell separation agent to the second culture vessel and to arrange and handle the culture surface of the second culture vessel parallel to the bottom surface of the main working portion; (d-6) placing the second culture vessel in the constant temperature culture portion for a predetermined time; (d-7) driving the culture vessel handler to add the culture solution to the second culture vessel and to arrange and handle the culture surface of the second culture vessel parallel to the bottom surface of the main working portion; (d-8) controlling to divide the cells remaining in the second culture vessel into a third tube; (d-9) placing the third tube in a centrifuge, driving the centrifuge to spin down, and removing the supernatant using a tip; And (d-10) moving the lid of the third tube to the mounting portion of the main work unit.

In one embodiment of the present invention, the step (d), (d-11) after a predetermined time elapses after the step (d-5), the culture surface of the second culture vessel is the bottom surface of the main working part In a state arranged in parallel with the, may further comprise the step of applying a physical impact on the left and right of the culture surface.

According to the present invention as described above, it is possible to solve the problem that many workers are required for conventional cell culture and it is difficult to control the errors occurring between workers, and the same number of cells as the minimum number of people required by the increase in demand for cell therapy products is the same. It has the effect of culturing with quality.

Furthermore, the present invention can control and manage the entire process with a small number of people, and is free from contamination of cells by workers, thereby providing more efficient quality control.

1 is a configuration diagram schematically illustrating an automation system to which a process of the present invention is applied.
Figure 2 is a flow chart for schematically illustrating a cell culture method of an embodiment of the present invention.
Figure 3a is a flow chart for explaining in detail the cell thawing process of Figure 2, Figure 3b is an exemplary view for explaining the location of the system in which the process of Figure 3a is performed.
Figure 4a is a flow chart for explaining in detail the medium exchange and culture process of Figure 2, Figure 4b is an exemplary view for explaining the location of the system in which the process of Figure 4a is performed.
FIG. 5A is a detailed flowchart for describing a passaging process of FIG. 2, and FIG. 5B is an exemplary diagram for describing a location of a system in which the process of FIG. 5A is performed.
FIG. 6A is a detailed flowchart for explaining the cell collection process of FIG. 2, and FIG. 6B is an exemplary diagram for describing a location of a system in which the process of FIG. 6A is performed.

In order to fully understand the constitution and effects of the present invention, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be embodied in various forms and various changes may be made. However, the description of the present embodiment is provided to make the disclosure of the present invention complete, and to fully inform the person of ordinary skill in the art the scope of the present invention. In the accompanying drawings, for convenience of description, the size of the components is larger than the actual drawings, and the ratio of each component may be exaggerated or reduced.

Terms such as 'first' and 'second' may be used to describe various components, but the components should not be limited by the above terms. The term may only be used to distinguish one component from another. For example, a 'first component' may be referred to as a 'second component' without departing from the scope of the present invention, and similarly, a 'second component' may also be referred to as a 'first component'. Can be. In addition, singular forms also include plural forms unless the context clearly indicates otherwise. Unless otherwise defined, terms used in the embodiments of the present invention may be interpreted as meanings commonly known to those skilled in the art.

Hereinafter, a method for automating cell culture according to an embodiment of the present invention will be described with reference to FIGS. 1 to 6.

1 is a configuration diagram schematically illustrating an automation system to which a process of the present invention is applied.

As shown in the figure, the automation system of one embodiment of the present invention may include a constant temperature culture unit 1, the main working unit 2, the storage unit 3 and the control unit (4).

At both ends of the main working part 2, the moving rails 42 and 43 are disposed on the upper part or the lower part of the main working part 2, and connect the two and are orthogonal to the moving rails 42 and 43, respectively. The movable rail 44 may be disposed. The moving rail 44 is composed of an upper moving rail and a lower moving rail, respectively, and further includes a vertical rail movably connected between the upper moving rail and the lower moving rail, and a support portion to which the robot device is connected to the vertical rail. Connected support parts along the vertical rail may be configured to be movable.

The robot device has a sensor attached to two grip parts to lift or move a variety of devices (tubes, culture packs, culture vessels, etc.), or move the device (tubes, culture packs, culture vessels, etc.) In order to perform the operation of opening and closing the lid of), for example, a robot device may be used. The robot device is operated by the control of the control unit 4, and the detailed configuration thereof is as known in the art, so a detailed description thereof will be omitted.

In the description of one embodiment of the present invention, the culture vessel is a container capable of culturing cells therein, and may be made of plastic, glass, or metal, for example, a flask, tube, 6-well, or dish. . However, the present invention is not limited thereto, and various kinds of culture vessels may be used.

The robot device may be configured as a joint to move vertically or horizontally, move vertically in the chamber by a vertical rail, and move to a desired position in the chamber by moving rails 42, 43, and 44. something to do.

Although omitted for the sake of simplicity, it is obvious that the movable rails, which are also horizontally movable, may be further included in the incubation unit 1 and the storage unit 3, and the rails 41 and 45 in each chamber may be upper and lower rails. It is apparent that the robot device may be connected to the vertical rail, including a vertical rail connecting the same.

The front or left and right sides of the robot device may be provided with a recognition unit for recognizing the identification code. Correspondingly, an identification code may be provided on the front surface of each seating portion of the culture vessel support 10 of the constant temperature culture unit 1. That is, the culture vessel support 10 of the constant temperature culture unit 1 includes a plurality of seating portions, such as a study, and the front side of the seating portion identification code (for example, a barcode or QR code) to identify the culture vessel inside Is provided, by the recognition unit of the robot device will be able to recognize the process of the culture vessel and move the culture vessel.

According to the present invention as described above, it is possible to culture the cells in each order in the main working unit 2, and by the control unit 4 controls the process by which the incubation unit 1 is cultured in a large amount, Cell culture may be possible.

The robot apparatus of the constant temperature culture unit 1, the main working unit 2, and the storage unit 3 may be controlled and moved by the control unit 4, respectively. To this end, each robot device may be connected to the control unit 4 through a wireless or wired network.

In an embodiment of the present invention, for example, the control unit 4 is disposed independently of the constant temperature culture unit 1, the main working unit 2, and the storage unit 3, but is not limited thereto. The control unit 4 may be disposed inside any one of the constant temperature culture unit 1, the main working unit 2, or the storage unit 3.

The constant temperature culture unit 1, the main working unit 2, and the storage unit 3 may each form one enclosed space, and the constant temperature culture unit 1 and the main working unit 2 in one space. And the reservoir 3 may be formed. When the constant temperature culture unit 1, the main working unit 2, and the storage unit 3 each form one enclosed space, the robot apparatus of the constant temperature culture unit 1 is brought to the main working unit 2. The openings may be formed in the constant temperature culture unit 1 and the main working unit 2, respectively, and the openings through which the robot apparatus of the storage unit 3 enters the main working unit 2 may be formed in the storage unit ( 3) and the main working part 2, respectively, and an opening through which a tube forming a path is connected so that the medium of the storage part 3 and the like are provided to the main working part 2, respectively. And the main working part 2, respectively.

However, in one embodiment of the present invention has been described that the opening is formed so that the robot device of each chamber enters the neighboring chamber, but the present invention is not limited thereto, the robot device of any one chamber is moved only in the chamber. In order to transfer the mechanism to another chamber, openings and support portions are respectively formed between the chambers, and for example, the control portion 4 is formed between the incubation portion 1 and the main working portion 2. ) May control the robot device of the main working unit (2) to place the instrument and the robot device of the constant temperature culture unit (1) to lift the instrument from the support to move to the support.

The constant temperature culture part 1 may be provided with a culture vessel support 10. The culture vessel support 10 may be a culture vessel for cultivation, it may include a temperature control device (not shown) to maintain a suitable temperature (for example 37 ℃) for the culture. As described above, the culture vessel support 10 may be provided with a plurality of seating portion in which a plurality of culture vessels can be arranged, the front may be attached with an identification code for the recognition of the culture vessel. The identification code may be, for example, a barcode, but is not limited thereto. Various types of codes may be used.

The main working unit 2 includes a holder 20, a heating unit 21, a tube holder 22, a centrifuge 23, a microscope 24, a culture vessel handler 25, a culture vessel holder 26 and Tip holder 28 may be included. One side of the main working unit 2 may be provided with a space 27 for the worker to put his hand to work. To this end, a plurality of openings into which the hand can be inserted may be provided on the front surface of the main working part 2.

The cradle 20 is a space for taking out the cell treatment agent having completed the process to the outside of the main work part 2, and a door (not shown) in an area corresponding to the cradle 20 of the housing of the main work part 2. ) May be formed.

The heating unit 21 may apply heat to thaw the cells. The heating unit 21 may be configured in a block shape, and a groove corresponding to the shape of the vial is formed, and the vial is inserted into the vial to defrost for a predetermined time.

The tube holder 22 is an area for mounting a plurality of tubes, and a space for opening the lid of the tube to be mounted on the side thereof may be formed. The supernatant may be removed from the tube centrifuged by the centrifuge 23 or may be used for dispensing the same amount of cell solution into a new tube.

Tip holder 28 is a region in which a plurality of sterilized tips are disposed, a plurality of tips may be disposed so that the robot device can take the tip for tipping.

The centrifuge 23 can spin down the cells in the tube under the control of the controller 4. At this time, in order to prevent dust, the cover may be disposed as a whole on the top of the centrifuge (23). Inside the centrifuge 23, an imaging device may be further arranged to identify the cell pellet and the supernatant of the tube to spin down. Images of the cell pellets and the supernatant taken by the image taking unit (not shown) may be provided to the controller 4.

The microscope 24 acquires an image for confirming the density and shape of the cells in the culture surface of the culture vessel moved from the incubation unit 1, and the image taken from the microscope 24 is transferred to the controller 4. It may be provided, the control unit 4 can check the culture degree of the cell. To this end, the control unit 4 may store in advance a reference image for checking the density and shape of the cells.

The culture vessel handler 25 may be rotated at a specific angle with the plurality of culture vessels fixed by the control of the controller 4. That is, the controller 4 washes the culture vessel, or the culture vessel handler 25 shakes the culture vessel from side to side, or rocking back and forth, so as to evenly apply a specific substance to the culture surface of the culture vessel. Or it may be controlled to shake the culture vessel to achieve a certain angle with the bottom surface, in the present specification, it will be used to mean 'handling'.

In this case, the culture vessel handler 25a provided in one region of the main work unit 2 may be configured to handle the culture surface of the culture vessel parallel to the bottom surface of the main work unit 2 and to handle the other region. The culture vessel handler 25b provided may be configured to handle the culture surface of the culture vessel perpendicularly to the bottom surface of the main working part 2. However, this is merely exemplary, and the present invention is not limited thereto, and the culture vessel handler 25 may be configured in the main working unit 2 in various ways. Alternatively, both culture vessel handlers 25 in both areas may be handled by arranging the culture vessel in parallel with the bottom surface of the main work unit 2, or vertically disposed.

Storage unit 3 is provided with a shelf 30, a side of the storage unit 3 may be provided with a space 31 for the operator to put his hand to work. To this end, a plurality of openings into which the hand can be inserted may be provided at the front of the storage unit 3. Shelf 30 may also be configured with a plurality of areas to store various objects.

The control unit 4 may be configured in the form of a server, or may be configured in the form of a terminal. The operator may check the process of an embodiment of the present invention through a display provided to the control unit 4, or may set the process work through the display.

Figure 2 is a flow chart for schematically illustrating a cell culture method of an embodiment of the present invention.

As shown in the figure, the method of one embodiment of the present invention, by thawing the frozen cells to wash the cryopreservation solution buried in the cells, transfer to a predetermined culture culture vessel to grow evenly attached on the bottom of the culture surface The cell thawing process may be performed (S10).

Figure 3a is a flow chart for explaining in detail the cell thawing process of Figure 2, Figure 3b is an exemplary view for explaining the location of the system in which the process of Figure 3a is performed.

First, the worker may thaw the cells by placing a plurality of vials containing the cells in a frozen state in the heating unit 21 (S11). In this case, the heating temperature of the heating unit 21 may be maintained at 37 ° C., for example, a heating unit may be provided inside or under the heating unit 21.

Then, from the tube connected from the storage unit 3, the control unit 4 fills the washing liquid through the tubing (tubing) to the tube disposed on the tube holder 22, and then adds a predetermined amount of thawed cells to the washing liquid. Can be washed (S12). In this case, the thawed cells may be added by tipping or pipetting by lifting the tip mounted on the tip holder 28 using a robot device.

After placing the tube in the centrifuge (23) and spin down the cells (S13), the control unit 4 again removes the supernatant using the tip leaving only the cell pellet (pellet) in the tube holder 22, The culture medium may be added to the tube containing the pellet using the tip (S14). Cells may be homogeneously suspended by the added culture. To this end, an empty storage tank is disposed in the storage unit 3, and may be stored and transferred to the supernatant through tubing. In this case, the type of the culture solution may be determined according to the cells to be cultured, and is not particularly limited.

Subsequently, the control unit 4 moves the culture vessel from the shelf 30 of the storage unit 3 to the one of the culture vessel holder 26 by using a robot device, and then dispenses a predetermined amount of the culture solution through the tubing, S14. In the culture medium is added, the suspended cell solution may be dispensed into a predetermined amount of the corresponding culture vessel using the tip (S15).

Subsequently, the control unit 4 moves the culture vessel to the culture vessel handler 25b, and arranges the culture surface of the culture vessel to be perpendicular to the bottom surface of the main working portion 2, so that the cells and the culture solution are uniformly mixed. Handle by shaking repeatedly from side to side or back and forth (S16), and then moved back to the culture vessel handler (25a), the culture surface of the culture vessel is placed in parallel with the bottom surface of the main working part (2), so that the cells The mixed culture can be handled to spread evenly (S17). The handling in S16 is for mixing and homogenizing the cells in the culture, and the handling in S17 is to shake in an orbital rotator manner, to spread the culture evenly over the culture surface of the culture vessel.

Thereafter, the control unit 4 may control the robot apparatus of each chamber to move and mount the culture vessel disposed on the culture vessel handler 25a to the culture vessel support 10 of the constant temperature culture unit 1 (S18). . At this time, the culture vessel may be mounted so that the culture surface in the culture vessel support 10 is parallel to the bottom surface of the constant temperature culture unit (1).

As described above, after the cell thawing process is completed and cultured in the incubation unit 1 for a predetermined time, the medium exchange and culturing process for supplying fresh medium to the cells being cultured in the culture culture vessel can be performed (S20). That is, the medium exchange and culturing process is a process of supplying a fresh medium to the cells in culture in the culture culture vessel.

Figure 4a is a flow chart for explaining in detail the medium exchange and culture process, Figure 4b is an exemplary view for explaining the location of the system in which the process of Figure 4a is performed.

As shown in the figure, the control unit 4 may move the culture vessel mounted on the culture vessel support 10 to the microscope 24, to take an image that can confirm the density and shape of the cells (S21). The image photographed by the microscope 24 may be provided to the controller 4, and the controller 4 compares the image transmitted from the microscope 24 with the reference image for determining the density and shape of the pre-stored cells. The density and morphology of the cells can be determined. Alternatively, the controller 4 may determine the density and the shape of the cell by calculating a ratio of the area occupied by the cell without comparing with the reference image. If the control unit 4 does not satisfy the density and shape of the predetermined cell, the control vessel will be able to return to the culture vessel support 10 of the incubation unit (1).

Thereafter, by moving the culture vessel to the culture vessel handler 25, the control unit 4 may open the lid of the culture vessel through the robot apparatus to remove the culture solution (S22), and dispense a predetermined amount of new culture solution through tubing. (S23).

Thereafter, the lid of the culture vessel is closed and the culture surface of the culture vessel in the culture vessel handler 25 is disposed and handled in parallel with the bottom surface of the main working part (2), and then the constant temperature culture unit (1) It can be controlled to move to the culture vessel support 10 (S25).

Such a medium exchange and culture process may be repeated a plurality of times after the completion of the culture in the incubation (1) for a predetermined time.

Subsequently, the control unit 4 may complete the medium exchange and culturing, and after culturing for a predetermined time in the incubation unit 1, may carry out a subculture process, which is a process of transferring the sufficiently cultured cells to another culture vessel. (S30). In the subculture, cells can be separated, and the cells can be cultured by dispensing the cells in a culture vessel filled with fresh medium.

FIG. 5A is a detailed flowchart for describing a passaging process of FIG. 2, and FIG. 5B is an exemplary diagram for describing a location of a system in which the process of FIG. 5A is performed.

The control unit 4 takes out the culture vessel from the culture vessel support 10 of the incubation unit 1 using a robotic device, and photographs an image with a microscope 24 to check the density and shape of the cells. The shape can be confirmed (S31). If the specified cell density and shape are satisfied, the controller 4 may drive the robot device and the culture vessel handler 25 to open the lid of the culture vessel and remove the culture solution (S32). However, if the density and the shape of the cells specified in S31 is not satisfied, the incubation unit 1 may be further incubated for a predetermined time.

Then, the control unit 4 is added to the washing liquid through the tubing and handled by shaking from side to side in a state in which the culture surface of the culture vessel is arranged parallel to the bottom surface of the main working portion 2, the culture surface of the culture vessel is the bottom surface After placing so as to be perpendicular to and can be repeated a plurality of times to open the lid of the culture vessel to remove the contents inside (S33). Thereby, the monolayer culture vessel can be washed. Handling in this process is for the purpose of rinsing the culture surface with a wash solution to wash off the remaining culture solution. Therefore, it will be possible to handle by shaking the culture vessel back and forth.

Thereafter, the control unit 4 may add a cell separation agent to the culture vessel washed in the culture vessel holder 26 (S34a), and move it to the culture vessel handler 25 so that the culture surface of the culture vessel is the bottom surface. The culture vessel handler 25 may be driven to be shaken from side to side or back and forth in a state in which it is arranged in parallel with each other (S34b). At this time, the cell separation agent may be added by tubing in the culture vessel holder (26). Handling in this process is to ensure that the cell separator is evenly applied to the culture surface. The cell separator may be, for example, an enzyme, and various materials capable of chemically separating cells may be used.

Thereafter, the control unit 4 may move the culture vessel to which the cell separation agent is added to the culture vessel support 10 of the constant temperature culture unit 1 (S35). When the cell is chemically separated from the culture vessel using a cell separation agent, an incubation is required for the reaction time, and for this, an incubation for a predetermined time may be required in the incubation unit 1.

After a predetermined reaction time has elapsed in the incubation unit 1, the controller 4 may move the culture vessel disposed on the culture vessel support 10 to the culture vessel handler 25.

Thereafter, the control unit 4 may apply a physical impact to the left and right of the culture surface in a state in which the culture surface of the culture vessel is disposed parallel to the bottom surface. This is a process of chemically separating the cells from the culture vessel through a cell separation agent, and physically separating the cells from the culture vessel through physical impact. However, physically separating the cells is not essential and may be omitted. In addition, the reaction time may vary depending on the type of cells, the type of culture vessel, and the type of cell separation agent.

Subsequently, the controller 4 may photograph and receive an image for confirming whether all the cells are separated from the culture surface using the microscope 24. If the cells do not fall out of the culture vessel, the above procedure can be repeated. The process of confirming the density and morphology of the cells using a microscope can also be optionally performed.

Then, the control unit 4 is added to the culture vessel in the culture vessel holder 26 through the tubing to the culture vessel (S36), the culture surface of the culture vessel is disposed so as to be parallel to the bottom surface and then shake left and right or back and forth to handle. The culture vessel handler 25 may be driven (S37). This is a process for neutralizing the cell separation agent, whereby the action of the cell separation agent can be inhibited. The handling in this process is to ensure that the cultures are evenly mixed with the cell separator so that the neutralization reaction occurs properly.

Thereafter, the controller 4 controls to dispense the cells remaining in the culture vessel to the tube mounted on the tube holder 22 (S38), and spins down in the centrifuge (S39a). After removing the remaining supernatant after leaving the cell pellet in the tube holder 22 again, the remaining cell pellet may be suspended in a culture solution to prepare a cell suspension (S39b). At this time, the removal of the culture solution may be performed by the tip.

Subsequently, the controller 4 adds the culture solution to the mixed container (for example, the culture container) through the tubing in the culture container handler 25, and then adds the cell suspension of the culture container using a tip to make the cells float. In addition, the culture vessel handler 25 may be driven to handle in a state in which the mixing vessel is disposed to be perpendicular to the bottom surface so that the solution is evenly mixed (S39c). Handling in this step is for producing a homogeneous suspension by mixing the culture and the cell suspension evenly.

Thereafter, the control unit 4 may tubing the culture solution to a new culture vessel, and may divide the cell suspension (suspension) suspended in S39c using the tip (S39d). In this case, when the suspension is dispensed into a new culture vessel, bubbles are generated, and the handle can be handled by appropriately adjusting the angle and speed of the handling so as not to generate bubbles in the suspension, and then the culture vessel support 10 of the incubation unit 1. It may be moved to (S39e).

As described above, when the subculture is completed and a predetermined time elapses, the controller 4 may repeatedly perform the medium exchange and the culture several times for the culture vessel containing the culture solution.

In addition, the control unit 40 may be repeatedly performed subculture several times using a multi-layer culture vessel containing the culture medium after a predetermined time after the medium exchange and culturing is performed. Such medium exchange and passaging may be performed several times depending on the type of cells to be cultured.

When passage is completed and a predetermined time has elapsed, the medium exchange and culture (S40) is once again performed, and then the cell collection process may be performed (S50). The cell collection process refers to harvesting cells in a pellet state by washing the cells in which the process is completed and treating the cell separation agent to separate the cells from the culture surface, and sufficiently washing the culture solution remaining on the cells.

FIG. 6A is a detailed flowchart for explaining the cell collection process of FIG. 2, and FIG. 6B is an exemplary diagram for describing a location of a system in which the process of FIG. 6A is performed. For convenience of description and the simplification of the drawings, the descriptions similar to those described with reference to FIGS. 5A and 5B are omitted in FIGS. 6A and 6B.

As shown in the figure, in the culture method of an embodiment of the present invention, the control unit 4 drives the robot apparatus to take out the culture vessel from the culture vessel support 10 of the constant temperature culture unit 1, the microscope 24 The density and the shape of the cells can be confirmed from the image taken with (S51). Thereafter, the control unit 4 may open the lid of the multilayer culture vessel in the culture vessel handler 25 and remove the culture solution (S52).

Then, the washing solution is added through the tubing, and the culture surface of the culture vessel is parallel to the bottom surface and shakes from side to side or back and forth to handle the mixing of the washing solution, and the culture surface of the culture vessel is perpendicular to the bottom surface and then cultured Opening the lid of the container can be repeated a plurality of times to remove the washing solution inside (S53). Thereby, the culture vessel can be washed. Handling in this process is for the purpose of rinsing the culture surface with a wash solution to wash off the remaining culture solution. Therefore, it will be possible to handle by shaking the culture vessel back and forth.

Thereafter, the control unit 4 may add a cell separation agent to the culture vessel washed in the culture vessel holder 26, and move it to the culture vessel handler 25 so that the culture surface of the culture vessel is parallel to the bottom surface. The culture vessel handler 25 may be driven to handle by shaking from side to side or back and forth in the disposed state. At this time, the cell separation agent may be added by tubing in the culture vessel holder (26). Handling in this process is to ensure that the cell separator is evenly applied to the culture surface. The cell separator may be, for example, an enzyme, and various materials capable of chemically separating cells may be used.

Thereafter, the control unit 4 may move the culture vessel to which the cell separation agent is added to the culture vessel support 10 of the incubation unit 1. When the cell is chemically separated from the culture vessel using a cell separation agent, an incubation is required for the reaction time, and for this, an incubation for a predetermined time may be required in the incubation unit 1.

After a predetermined reaction time has elapsed in the incubation unit 1, the controller 4 may move the culture vessel disposed on the culture vessel support 10 to the culture vessel handler 25.

Thereafter, the control unit 4 may apply a physical impact to the left and right of the culture surface in a state in which the culture surface of the culture vessel is disposed parallel to the bottom surface. This is a process of chemically separating the cells from the culture vessel through a cell separation agent, and physically separating the cells from the culture vessel through physical impact. However, physically separating the cells is not essential and may be omitted. In addition, the reaction time may vary depending on the type of cells, the type of culture vessel, and the type of cell separation agent.

Subsequently, the controller 4 may photograph and receive an image for confirming whether all the cells are separated from the culture surface using the microscope 24. If the cells do not fall out of the culture vessel, the above procedure can be repeated. The process of confirming the density and morphology of the cells using a microscope can also be optionally performed.

Thereafter, the controller 4 adds the culture solution to the culture vessel through the tubing in the culture vessel holder 26 (S54a), and arranges the culture surface of the culture vessel in parallel with the bottom surface in the culture vessel handler 25. In the culture vessel handler 25 can be driven to shake the culture vessel from side to side or back and forth (S54b). This is a process for neutralizing the cell separation agent, whereby the action of the cell separation agent can be inhibited. The handling in this process is to ensure that the cultures are evenly mixed with the cell separator so that the neutralization reaction occurs properly. In addition, the culture solution may be replaced with a neutralizing agent.

Thereafter, the controller 4 may control the cells remaining in the culture vessel to be dispensed to the tube mounted on the tube holder 22 using the tip (S55), and spin down in the centrifuge (S56). . Thereafter, the cell suspension may be left in the tube holder 22 and the supernatant may be removed using the tip (S57). In this case, while the spin down is performed in the centrifuge 23, an image of a tube in which the spin is being progressed may be acquired through the image photographing apparatus inside the centrifuge 23, and the controller 4 may thereby perform a spin down. You can check whether it is completed.

Thereafter, the control unit 4 adds the washing solution through the tubing to the cell pellet from which the culture solution has been removed (S58), and puts a part of the solution into another new tube to the mounting unit 20, so that the worker has a main working unit ( Cell counting may be performed outside of 1) (S59).

When the cell counting is completed, the controller 4 may spin down the final tube through the centrifuge 23, and remove the remaining supernatant through the tubing, leaving only the cell pellet in the tube holder 22.

The cell pellets finally left as such may be moved to the holder 20 in a closed state of the tube, and may be provided to the worker (S60).

It is apparent that in one embodiment of the present invention, the material most produced may vary depending on the material initially provided. In other words, depending on the type of wash solution used in the middle of the process and the type and differentiation of the culture medium may be variously changed. Therefore, in one embodiment of the present invention is not limited to the type of wash solution and the type of culture medium, various kinds of wash solution and culture solution may be used according to the material and the type of cells to be harvested.

Although embodiments according to the present invention have been described above, these are merely exemplary, and it will be understood by those skilled in the art that various modifications and equivalent embodiments of the present invention are possible therefrom. Therefore, the true technical protection scope of the present invention will be defined by the following claims.

1: constant temperature culture unit 2: main working unit
3: storage unit 4: control unit

Claims (10)

In a system comprising a storage unit, a main working unit, a constant temperature culture unit and a control unit, in the method for culturing cells under the control of the control unit,
(a) thawing the vial containing the frozen cells, dispensing in a first culture vessel and culturing in the incubation;
(b) moving the first culture vessel cultured in the incubation portion to the main working portion, exchanging a culture solution, and culturing in the incubation portion;
(c) separating the cells from the culture surface of the first culture vessel, aliquoting the second culture vessel, and culturing in the incubation portion; And
(d) separating the cells from the culture surface of the second culture vessel and collecting the cells.
According to claim 1, wherein the step (a),
(a-1) placing the vial containing the frozen cells into a heating unit of the main work unit;
(a-2) adding washing solution to the first tube through tubing, and adding the thawed cells from the vial to the first tube with a tip;
(a-3) placing the first tube in a centrifuge, driving the centrifuge to spin down, and removing the supernatant using a tip;
(a-4) adding a culture solution to the first tube through a tip to prepare a cell solution by mixing the cell pellet and the culture solution;
(a-5) dispensing the culture solution through the tubing in the first culture vessel;
(a-6) dispensing the cell solution into the first culture vessel;
(a-7) driving the culture vessel handler to arrange and handle the culture surface of the first culture vessel in a state perpendicular to the bottom surface of the main working portion;
(a-8) driving the culture vessel handler to arrange and handle the culture surface of the first culture vessel in parallel with the bottom surface of the main working portion; And
(A-9) Cell culture automation method comprising the step of moving the first culture vessel to the incubation.
The method of claim 2, wherein the culture solution is stored in a predetermined container in the storage unit and provided to the first tube or the first culture container of the main working unit through a tube from the predetermined container.
According to claim 1, wherein step (b),
(b-1) driving the culture vessel handler to remove the culture solution in the first culture vessel;
(b-2) dispensing a new culture solution through tubing to the first culture vessel; And
(b-3) driving the culture vessel handler to arrange and handle the culture surface of the first culture vessel in a state parallel to the bottom surface of the main working portion.
The method of claim 4, wherein step (b) comprises:
(b-4) receiving the image of the culture surface of the first culture vessel from the microscope further comprising the step of confirming the density and shape of the cells of the cells.
The method of claim 5, wherein the density and shape of the cells are determined by comparing a reference image and an image received from the microscope, or by calculating a ratio of the area occupied by the cells.
The method of claim 1, wherein step (c) comprises:
(c-1) driving the culture vessel handler to remove the culture solution in the first culture vessel;
(c-2) adding the washing liquid to the first culture vessel through tubing;
(c-3) driving the culture vessel handler to arrange and handle the culture surface of the first culture vessel parallel to the bottom surface of the main working portion;
(c-4) driving the culture vessel handler to remove the washing solution in the first culture vessel;
(c-5) adding a cell separator to the first culture vessel, and driving the culture vessel handler to arrange and handle the culture surface of the first culture vessel parallel to the bottom surface of the main working portion;
(c-6) placing the first culture vessel in the constant temperature culture portion for a predetermined time;
(c-7) driving the culture vessel handler to add the culture solution to the first culture vessel and to arrange and handle the culture surface of the first culture vessel parallel to the bottom surface of the main work unit;
(c-8) dispensing the cells remaining in the first culture vessel in a second tube;
(c-9) arranging the second tube in a centrifuge, driving the centrifuge to spin down, removing the supernatant using a tip, and floating the remaining cell pellet in a culture medium to prepare a cell suspension;
(c-10) adding a culture solution to the mixing vessel through tubing, and adding the cell suspension of the first culture vessel to the mixing vessel using a tip to prepare a suspension solution;
(c-11) driving the culture vessel handler to arrange and handle the culture surface of the mixing vessel perpendicular to the bottom; And
(C-12) Dispensing the suspension of the mixed vessel to the second culture vessel and cell culture automation method comprising the step of moving to the incubation.
The method of claim 7, wherein step (c) is
(c-13) After a predetermined time has elapsed after the step (c-5), the culture surface of the first culture vessel is arranged to be parallel to the bottom surface of the main work portion, and physically on the left and right sides of the culture surface. Cell culture automation method further comprising the step of impacting.
The method of claim 1, wherein step (d)
(d-1) driving the culture vessel handler to remove the culture solution in the second culture vessel;
(d-2) adding the washing liquid to the second culture vessel through tubing;
(d-3) driving the culture vessel handler to arrange and handle the culture surface of the second culture vessel parallel to the bottom surface of the main working portion;
(d-4) driving the culture vessel handler to remove the washing solution in the second culture vessel;
(d-5) driving the culture vessel handler to add the cell separation agent to the second culture vessel and to arrange and handle the culture surface of the second culture vessel parallel to the bottom surface of the main working portion;
(d-6) placing the second culture vessel in the constant temperature culture portion for a predetermined time;
(d-7) driving the culture vessel handler to add the culture solution to the second culture vessel and to arrange and handle the culture surface of the second culture vessel parallel to the bottom surface of the main working portion;
(d-8) controlling to divide the cells remaining in the second culture vessel into a third tube;
(d-9) placing the third tube in a centrifuge, driving the centrifuge to spin down, and removing the supernatant using a tip; And
(d-10) Cell culture automation method comprising the step of moving to the holder of the main work in the closed state of the third tube.
The method of claim 9, wherein step (d)
(d-11) After a predetermined time has elapsed after the step (d-5), the culture surface of the second culture vessel is arranged to be parallel to the bottom surface of the main working portion, and the physical surface is formed on the left and right sides of the culture surface Cell culture automation method further comprising the step of impacting.

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