JP7238512B2 - Subculture auxiliary device and subculture operation method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a subculturing assisting device and a subculturing operation method for performing a detachment step when subculturing adherent cells respectively housed in a plurality of housing sections.

In the process of culturing adherent cells, an operation called passaging is performed when maintaining the cells for a long period of time or increasing the number of cells (see, for example, Patent Document 1 below). Passaging is the operation of transferring cells in culture to a new container, and is performed when, for example, the cells cover the bottom surface of the culture container and there is no place to adhere.

When subculturing the adherent cells, a detachment process for recovering the cells from the container is performed as a preliminary step. The detachment process includes, for example, a culture supernatant removal process, a washing process, a reagent reaction process, a medium supply process, a medium agitation process, and a suspension recovery process, and is carried out according to the following procedure.

Adherent cells to be cultured adhere to the bottom surface of the container. In this state, first, the culture supernatant is removed from the storage section (culture supernatant removal step), so that only cells are left in the storage section. After that, a washing liquid is supplied to the storage section, and the cells are washed by removing the washing liquid after a predetermined time has passed (washing step). Then, a specific reagent is supplied to the container, and the reagent is removed after a predetermined period of time has elapsed, thereby cutting the binding of cell adhesion proteins to the bottom surface of the container (reagent reaction step).

In this way, after the binding of the cell adhesion protein to the bottom surface of the container is cut, the medium is supplied to the container (medium supply step), and then the medium in the container is agitated (medium agitation step), the cells are peeled off from the bottom surface of the container, and a suspension in which the cells are mixed in the medium is produced. Then, the suspension is recovered from the storage section (suspension recovery step), and the suspension is transferred to another storage section.

Cells are accommodated in a plurality of storage sections, respectively, and when passage work is performed on the culture medium in each storage section, generally, the steps described above are sequentially performed on the plurality of storage sections. FIG. 5A is a conventional example of a timing chart when performing a culture supernatant removal process, a washing process, and a reagent reaction process for a plurality of storage units. FIG. 5B is a conventional example of a timing chart when performing a culture medium supply process, a culture medium agitation process, and a suspension collection process for a plurality of storage units.

As shown in FIG. 5A, the culture supernatant removal step is sequentially performed on each storage section, and after the culture supernatant removal step is completed on all storage sections, the washing process is sequentially performed on each storage section. . The cleaning process includes a cleaning liquid supply process of supplying cleaning liquid to each container and a cleaning liquid removing process of removing cleaning liquid from each container after a predetermined time has elapsed. The cleaning liquid supply process is sequentially performed for each storage section, and after the cleaning liquid supply process for all the storage sections is completed, the cleaning liquid removal process is sequentially performed for each storage section. Then, the reagent reaction process is started in order from the storage unit for which the cleaning process has been completed. The reagent reaction process is performed in parallel in each container.

When the reagent reaction step is completed in each container, as shown in FIG. 5B, the medium supply step is sequentially performed for each container. The medium agitation step is performed sequentially. Then, after the culture medium agitating step for all the containing parts is completed, the suspension collecting process is sequentially performed for each containing part.

WO2015/166845

Cell culture is performed in an incubator in which the temperature is kept constant. During subculturing, the cultured cells are moved out of the incubator and subjected to the detachment process as described above, followed by subculturing. As a result, there is a problem that the temperature inside the container decreases during the detachment process, and the survival rate of the cells decreases. Furthermore, when the temperature drops, it takes a long time to reach the required temperature after returning to the incubator. was a major cause of decreased survival.

In addition, when the reagent reaction step is completed in each container, the binding of cell adhesion proteins to the bottom surface of each container is cut. At the same time, the cells adhere to the bottom surface of each container again. As described above, when the medium supply step is sequentially performed for each container, and after the medium supply step for all the containers is completed, the medium stirring step is sequentially performed for each container. As indicated by the dotted line in 5B, a waiting time occurs between the supply of the culture medium to each container and the start of the medium stirring process for each container. During this waiting time, the cells re-adhered to the bottom surface of each container, so when the medium stirring step was performed after that, the re-adhered cells were forcibly peeled off, and as a result, the cells There was a problem that the survival rate of In addition, as shown by the dotted line in FIG. 5B, the waiting time in each container is different, which causes a difference in the influence on each container, which is one of the factors that cause variations in culture results. . The length of the dotted line in FIG. 5B means the length of the waiting time, that is, the length of the immersion time (standing time) of each container.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a subculturing assisting device and a subculturing operation method that can improve cell viability and reduce variations in culture results. .

(1) A passaging assisting device according to the present invention is a passaging assisting device for executing a detachment step when performing a passaging operation on cells respectively accommodated in a plurality of storage units, and is characterized by: A mechanism, a container holding section, a temperature control mechanism, a culture supernatant removal process execution section, a reagent reaction process execution section, a medium supply process execution section, a medium agitation process execution section, and a suspension collection process execution section. and

The dispensing mechanism dispenses and drips the solution. The container holding part holds a container for culturing cells. The temperature control mechanism controls the temperature of the container. The culture supernatant removal step executing unit executes a culture supernatant removal step of removing the culture medium from each of the storage units while leaving the cells. The reagent reaction step execution unit supplies a reagent to each container after the culture supernatant removal step has been performed, and removes the reagent after a predetermined period of time has elapsed, thereby binding cell adhesion proteins to each container. A reagent reaction step for cleaving is performed. The culture medium supply step execution unit performs a culture medium supply step of supplying a culture medium to each container after the reagent reaction step has been performed. The medium agitating process execution unit performs a medium agitating process of generating a suspension in which cells are mixed in the medium by agitating the medium in each container after the medium supply process is performed. The suspension collection process execution unit executes a suspension collection process of collecting suspension from each container after the medium stirring process is executed. The temperature control mechanism heats the interior of each container to a temperature at which the cell activity is maintained and the reagent reacts appropriately, at least while the reagent reaction process execution unit is executing the reagent reaction process.

According to such a configuration, at least during the time the reagent reaction step is being performed, the inside of each container is heated to a temperature at which the cell activity is maintained and the reagent reacts appropriately. can be prevented from declining. As a result, insufficient reaction of the reagent due to a temperature drop can be suppressed, thereby improving the viability of the cells. The temperature at which the cell activity is maintained and the reagent reacts appropriately is preferably 33 to 38°C, more preferably 37°C.

(2) In the subculture assisting device, at least the medium supplying step, the medium stirring step, and the suspension collecting step are performed in a series of steps for each of the storage units, so that the series of steps are performed individually. It is preferably performed sequentially for the receptacles.

According to such a configuration, at least the medium supply step, the medium agitation step, and the suspension collection step are executed in a series of steps for each container. After the supply, no waiting time occurs until the medium agitating process is started for each container. This can prevent cells from re-adhering to the bottom surface of each container after the medium is supplied to each container. Therefore, when the medium agitating step is performed, the cells that have re-adhered to the bottom surface of each container are not forcibly detached, thereby improving the survival rate of the cells.

(3) The subculture assisting device may further include a washing process execution unit. The washing process execution unit supplies a washing liquid to each container after the culture supernatant removal process has been executed, and removes the washing liquid after a predetermined time has elapsed, thereby executing a washing process for washing cells. The reagent reaction step supplies a reagent to each container after the washing step has been performed. In this case, it is preferable that at least the culture supernatant removing step and the washing step are performed in a series of steps for each container, such that the series of steps are sequentially performed for each container.

According to such a configuration, since at least the culture supernatant removal step and the washing step are performed in a series of steps for each container, the predetermined time in the subsequent reagent reaction step is individually set for each container. can be managed. As a result, variations in the predetermined time can be suppressed.

(4) In the subculture assisting device, it is preferable that the series of steps for each storage section is performed sequentially without waiting time for each storage section.

According to such a configuration, a series of steps that are sequentially performed for each accommodating portion are performed without waiting time in between, so that the time required for the peeling step can be shortened. In addition, it is possible to make the contents of the work steps performed for each storage part the same in terms of time, and to suppress variations in the effect of each storage part on the cells.

(5) A passage operation method according to the present invention is a passage for performing a detachment step when performing passage work on cells housed in a plurality of housing units using the passage auxiliary device. This alternative operation method includes a culture supernatant removal step, a reagent reaction step, a medium supply step, a medium stirring step, a suspension recovery step, and a temperature control step.

In the culture supernatant removing step, the culture medium is removed from each of the plurality of storage sections while leaving the cells. In the reagent reaction step, a reagent is supplied to each container after the culture supernatant removal step is performed, and the reagent is removed after a predetermined time has elapsed, thereby severing the binding of cell adhesion proteins to each container. Let In the medium supply step, the medium is supplied to each container after the reagent reaction step has been performed. In the medium agitation step, a suspension of cells mixed in the medium is generated by agitating the medium in each container after the medium supply step is performed. In the suspension recovery step, the suspension is recovered from each container after the medium stirring step is performed. In the temperature control step, the interior of each container is heated to a temperature at which cell activity is maintained and reagents react appropriately, at least during the time the reagent reaction step is being performed.

(6) In the subculture operation method, at least the medium supplying step, the medium stirring step, and the suspension collecting step are performed in a series of steps for each of the storage units, so that the series of steps are performed individually. It is preferably performed sequentially for the receptacles.

(7) The subculture operation method may further include a washing step. In the washing step, the cells are washed by supplying a washing liquid to each container after the culture supernatant removal step is performed, and removing the washing liquid after a predetermined time has elapsed. In the reagent reaction step, a reagent is supplied to each container after the washing step has been performed. In this case, it is preferable that at least the culture supernatant removing step and the washing step are performed in a series of steps for each container, such that the series of steps are sequentially performed for each container.

(8) In the subculture operation method, it is preferable that the series of steps for each container is performed sequentially without waiting time for each container.

According to the present invention, it is possible to suppress insufficient reaction of the reagent due to a decrease in the temperature inside the container, thereby improving the viability of the cells. In addition, if at least the medium supply step, the medium agitation step, and the suspension recovery step are performed in a series of steps for each container, the medium can be re-adhered to the bottom surface of each container when the medium agitation step is performed. Since the cells are not forcibly detached, the survival rate of the cells is improved.

BRIEF DESCRIPTION OF THE DRAWINGS It is the block diagram which showed schematic structure of the subculture assistance apparatus which concerns on one Embodiment of this invention. 1. It is a figure for demonstrating the specific operation|movement in each process performed by the succession assistance apparatus of FIG. 1. It is a figure for demonstrating the specific operation|movement in each process performed by the succession assistance apparatus of FIG. 4 is a timing chart when performing a culture supernatant removal process, a washing process, and a reagent reaction process for a plurality of storage units. 4 is a timing chart when performing a medium supply process, a medium agitation process, and a suspension recovery process for a plurality of storage units. It is a conventional example of a timing chart when performing a culture supernatant removal process, a washing process, and a reagent reaction process for a plurality of storage units. It is a conventional example of a timing chart when performing a culture medium supply process, a culture medium agitation process, and a suspension recovery process for a plurality of storage units. FIG. 10 is a diagram showing the relationship between the standing time from the supply of the medium to the storage section to the start of the medium agitating process after the bonding of the adhesive protein of the cells to the bottom surface of the storage section is cut, and the survival rate of the cells. is. When the temperature of the container is adjusted to 37°C during the reagent reaction process (plate heating), and when there is a change in temperature as in the conventional culture process (control), the measured temperature It is a figure which showed a time-dependent change. FIG. 7B shows the cell viability in each case of FIG. 7A.

1. Schematic Configuration of Subculture Auxiliary Device FIG. 1 is a block diagram showing a schematic configuration of a subculture auxiliary device according to an embodiment of the present invention. This subculturing assisting device is a device for performing a detachment step when subculturing cells 21 housed in a plurality of housing units 1 .

The plurality of housing portions 1 are configured by a plurality of recesses formed on the upper surface of the plate member 3. As shown in FIG. Although three housings 1 are shown in FIG. 1, the number of housings 1 is arbitrary. In addition, the structure is not limited to one plate member 3 in which a plurality of storage portions 1 are formed, and an individual plate member may be prepared for each storage portion 1 .

Cells are cultured in the culture supernatant 2 in each container 1 . During cell culture, the dish member 3 is placed in an incubator (not shown). Then, when the cells 21 need to be subcultured, the dish member 3 is taken out from the incubator and set in the subculture assisting device according to the present embodiment.

This passage assisting device is provided with a temperature control mechanism 4 for controlling the temperature of the dish member 3 . The dish member 3 taken out of the incubator is set in the temperature control mechanism 4 and is temperature-controlled at an appropriate temperature. In this embodiment, the temperature control mechanism 4 constitutes a container holder that holds the dish member 3, which is a container for culturing cells. However, the temperature control mechanism 4 may be configured separately from the container holding portion. The temperature control mechanism 4 is equipped with a heater (not shown) for heating the dish member 3 . By controlling the driving of this heater, the inside of each housing portion 1 can be heated to a set predetermined temperature.

When subculturing, the detachment process performed by the subculture assisting device as a pre-step includes, for example, a culture supernatant removal process, a washing process, a reagent reaction process, a medium supply process, a medium agitation process, and a suspension recovery process. and so on. Each of these steps is performed for each container 1 while the plate member 3 is set in the temperature control mechanism 4 .

In each step as described above, an operation of supplying various liquids to and removing various liquids from each container 1 is performed. The liquid includes the culture supernatant 2 in which the cells 21 contained in each container 1 are immersed, as well as a new medium 5, a washing liquid 6, a reagent 7, and the like. The medium 5 , washing liquid 6 and reagent 7 are stored in advance in the liquid storage section 8 .

A syringe 9 is used to supply and remove the liquid to and from each container 1 . This syringe 9 constitutes a dispensing mechanism for dispensing and dripping the solution. The syringe 9 is provided with a connecting portion 91 , and a tip 92 is attached to and detached from the tip of the connecting portion 91 . The liquid can be sucked into the tip 92 by inserting the tip of the tip 92 into the liquid and performing a suction operation with the syringe 9 . Further, when the liquid is sucked into the chip 92 and an ejection operation is performed with the syringe 9, the liquid in the chip 92 can be ejected. A plurality of tips 92 are set in advance in the subculture assisting device, and the tip 92 at the tip of the connecting portion 91 is replaced as necessary. However, the dispensing mechanism is not limited to the configuration described above, and may have a configuration including a plurality of syringes 9, for example.

The passage auxiliary device is provided with a control unit 10 for controlling the operations of the temperature control mechanism 4, the syringe 9, and the like. The control unit 10 includes a CPU (Central Processing Unit). By the CPU executing a program, the control unit 10 performs a culture supernatant removal process execution unit 11, a washing process execution unit 12, a reagent reaction process execution unit 13, a medium supply process execution unit 14, a medium agitation process execution unit 15, It functions as a suspension collection process execution unit 16, a temperature control unit 17, and the like. The control unit 10 is not limited to the configuration provided in the subculture assisting device, and may be configured by a personal computer capable of communicating with the subculture assisting device.

2. Specific Operations in Each Step FIGS. 2 and 3 are diagrams for explaining specific operations in each step performed by the passage assisting device of FIG. 1 . Each step will be specifically described below with reference to FIGS. 1 to 3. FIG.

As shown in FIG. 2A, cells 21 cultured in the culture supernatant 2 adhere to the bottom surface of the container 1 . In this state, first, the culture supernatant removal step execution unit 11 removes the culture supernatant 2 from the storage unit 1 while leaving the cells 21, thereby executing the culture supernatant removal step. In the culture supernatant removing step, the syringe 9 is used to aspirate the culture supernatant 2 in the container 1 and is discharged to a waste liquid port or a waste liquid container (not shown). As a result, only the cells 21 are left in the container 1 (see FIG. 2B).

The washing process execution unit 12 performs processing for executing a washing process for washing the cells 21 left in the container 1 . In the washing step, the syringe 9 is used to supply the washing liquid 6 into the container 1 after the culture supernatant removing step has been performed (see FIG. 2C). After a predetermined period of time has elapsed, the washing liquid 6 in the storage section 1 is sucked using the syringe 9 and discharged to the waste liquid port or the waste liquid container. As a result, the washing liquid 6 in the container 1 is removed together with contaminants adhering to the cells 21 (see FIG. 2D), so that the cells 21 are washed.

As the washing liquid 6, for example, PBS (Phosphate Buffered Saline) is used. However, the washing liquid 6 is not limited to PBS and may be any other liquid as long as it is suitable for removing contaminants adhering to the cells 21 . It is also possible to omit the washing process.

The reagent reaction process executing unit 13 performs a process for executing the reagent reaction process on the cells 21 after the washing process has been executed. In the reagent reaction step, the syringe 9 is used to supply the reagent 7 into the container 1 after the washing step has been performed (see FIG. 3A). After a predetermined period of time has elapsed, the reagent 7 in the container 1 is aspirated using the syringe 9 and discharged to the waste liquid port or the waste liquid container. Until the predetermined time has passed, the action of the reagent 7 cuts the binding of the adhesive protein of the cell 21 to the bottom surface of the container 1 . Therefore, when the reagent 7 in the container 1 is drained (see FIG. 3B), the cells 21 are not adhered to the bottom surface of the container 1, or are almost not adhered. Here, the "unfixed state" means a state in which the molecular bonds of the adhesive protein are broken.

As the reagent 7, for example, a solution containing an enzyme such as trypsin (an enzyme solution such as a trypsin-EDTA solution) is used. Due to the action of this enzyme, the protein that adheres the cell 21 to the bottom surface of the container 1 can be cut. The predetermined time in the reagent reaction step is, for example, 6 to 12 minutes, more preferably about 10 minutes. However, the reagent 7 is not limited to the enzyme solution, and may be any other liquid as long as it is suitable for cleaving the binding of the adhesive protein of the cells 21 to the container 1 .

The culture medium supply process execution unit 14 performs processing for executing the culture medium supply process of supplying new culture medium 5 to the container 1 . In the medium supply step, the syringe 9 is used to supply the medium 5 into the container 1 after the reagent reaction step has been performed (see FIG. 3C). However, before supplying the new medium 5 to the storage part 1, the washing liquid 6 is supplied into the storage part 1, and after a predetermined time has passed, the washing liquid 6 in the storage part 1 is sucked with the syringe 9 and discharged. A washing step may be performed.

The medium agitation process execution unit 15 performs processing for executing the medium agitation process by agitating the medium 5 in the container 1 after the medium supply process has been executed. Cells 21 in storage section 1 are separated from the bottom surface of the storage section by the liquid flow caused by stirring in the medium agitation process because the bonding of the adhesive protein to storage section 1 has been cut in the reagent reaction step. Cells 21 are mixed to form a suspension (see Figure 3D).

The suspension collection process execution unit 16 performs processing for executing a suspension collection process of collecting suspension from the storage unit 1 after the medium stirring process has been executed. In the suspension collection step, the syringe 9 is used to suck the suspension in the container 1 . As a result, the suspension 22 is sucked into the chip 92 (see FIG. 3E), and the suspension 22 is transferred to another container.

The temperature control unit 17 controls the operation of the temperature control mechanism 4 to perform a temperature control process for heating the inside of the housing unit 1 to a predetermined temperature. The predetermined temperature is, for example, 33 to 38°C, more preferably 37°C. In this embodiment, the inside of the container 1 is continuously heated from the culture supernatant removing step to the medium stirring step. However, at least in the reagent reaction step, the inside of the containing portion 1 may be heated during the period from when the reagent 7 is supplied into the containing portion 1 until it is removed.

3. Execution Procedure of Each Process Before Reagent Reaction Process FIG. As described above, the cleaning process includes a process of supplying the cleaning liquid to each storage section 1 (cleaning liquid supply process) and a process of removing the cleaning liquid from each storage section 1 after a predetermined time (cleaning liquid removal process).

As shown in FIG. 4A, in the present embodiment, the culture supernatant removing step and the washing step are performed in a series of steps for each container 1 . That is, the process of continuously performing the culture supernatant removing step and the washing step for one container 1 and then continuously performing the culture supernatant removing step and the washing step for the next container 1 is It is repeatedly executed for a plurality of storage units 1 . As a result, a series of steps (the culture supernatant removal step and the washing step) before the reagent reaction step are sequentially performed for each container 1 .

A series of steps (culture supernatant removing step and washing step) for each container 1 before the reagent reaction step are sequentially performed without waiting time for each container 1 . That is, immediately after the washing process for one container 1 is completed, the culture supernatant removal process for the next container 1 is started without waiting time. Then, the reagent reaction process is started in order from the container 1 for which the washing process has been completed. The reagent reaction process is performed in parallel in each container 1 .

4. Execution Procedure of Each Step after Reagent Reaction Step FIG. 4B is a timing chart when performing the medium supply step, the medium stirring step, and the suspension collection step for the plurality of storage units 1 .

As shown in FIG. 4B, in the present embodiment, the medium supply step, the medium agitation step, and the suspension collection step are performed in a series of steps for each container 1 . That is, after continuously performing the medium supply step, the medium stirring step, and the suspension recovery step for one storage unit 1, the medium supply step, the medium stirring step, and the suspension recovery step for the next storage unit 1 A process of continuously executing the steps is repeatedly executed for a plurality of storage units 1 . As a result, a series of steps (medium supply step, medium agitation step, and suspension recovery step) after the reagent reaction step are sequentially performed for each container 1 .

A series of steps (medium supply step, medium agitation step, and suspension collection step) for each container 1 after the reagent reaction step are sequentially executed without waiting time for each container 1 . That is, immediately after the suspension recovery step for one container 1 is completed, the culture medium supply step for the next container 1 is started without waiting time.

5. Effect (1) In the present embodiment, at least during the time the reagent reaction step shown in FIGS. , the inside of each container 1 is heated to a temperature at which the activity of the cells 21 is maintained and the reagents 7 react appropriately, so that the temperature of the cells 21 and the reagents 7 can be prevented from dropping. As a result, insufficient reaction with the reagent 7 due to a temperature drop can be suppressed, and adhesion proteins of the cells 21 can be appropriately cleaved, thereby improving the viability of the cells 21 .

(2) In the present embodiment, as shown in FIG. 4B, at least the culture medium supply process, the culture medium agitation process, and the suspension collection process are performed in a series of steps for each container 1 . Therefore, after the culture medium 5 is supplied to each container 1 (see FIG. 3C), no waiting time occurs until the culture medium agitation process is started for each container 1 (see FIG. 3D). This can prevent the cells 21 from adhering again to the bottom surface of each container 1 after the medium 5 is supplied to each container 1 . Therefore, when the medium agitating step is performed, the cells 21 re-adhered to the bottom surface of each container 1 are not forcibly detached, so that the survival rate of the cells 21 is improved.

(3) In the present embodiment, as shown in FIG. 4A, at least the culture supernatant removal step and the washing step are performed in a series of steps for each container 1 . Therefore, it is possible to individually manage the predetermined time (the period from when the reagent 7 is supplied into each container 1 until it is removed) in the subsequent reagent reaction step for each container 1 . As a result, variations in the predetermined time can be suppressed.

(4) In the present embodiment, a series of steps (culture supernatant removal step and washing step in FIG. 4A, or medium supply step, medium stirring step and suspension collection in FIG. 4B) that are sequentially performed for each storage unit 1 process) is executed without waiting time in between, the time required for the peeling process can be shortened.

5. Experimental results on survival rate FIG. 6 shows the state after the bond of the adhesion protein of the cells 21 to the bottom surface of the container 1 is cut, and the medium is left to stand until the medium stirring step is started after the medium is supplied to the container 1. FIG. 4 is a diagram showing the relationship between time and survival rate of cells 21; According to the experimental results shown in FIG. 6, compared to the survival rate of the cells 21 when there is no standing time, the survival of the cells 21 when the medium stirring step is started after the medium is left to stand for 1 min. The viability decreased by 5.1%, and the survival rate of the cells 21 decreased by 9.6% when the medium agitation step was started after standing for 5 minutes after supplying the medium. This is because the cells 21 re-adhere to the bottom surface of the housing part 1 over time, and the stationary time is preferably as short as possible (for example, less than 40 seconds).

FIG. 7A shows a case where the container 1 is temperature-controlled at 37° C. during the reagent reaction step (plate heating) and a case where the temperature changes as in the conventional culturing operation (control). , respectively, showing temporal changes in the measured temperature. FIG. 7B is a diagram showing the viability of cells 21 in each case of FIG. 7A. As shown in FIG. 7A, when the container 1 is temperature-controlled, the temperature of the container 1 is kept within 2° C. As a result, as shown in FIG. It can be confirmed that this is an improvement over the past. As a result of performing a t-test on each data of the survival rate when the temperature of the housing unit 1 was adjusted (plate heating) and when there was a temperature change (control), p < 0.05, a significant difference It was confirmed that

6. Modifications In the above embodiment, the culture supernatant removal step and washing step in FIG. 4A and the medium supply step, medium stirring step, and suspension recovery step in FIG. We have described the configuration as it would be executed. However, the configuration is not limited to such a configuration, and a configuration in which only one of the steps is performed in a series of steps for each housing portion 1 may be employed.

1 storage unit 2 medium 3 dish member 4 temperature control mechanism 5 medium 6 washing liquid 7 reagent 8 liquid storage unit 9 syringe 10 control unit 11 culture supernatant removal process execution unit 12 washing process execution unit 13 reagent reaction process execution unit 14 medium supply process Execution unit 15 Medium stirring process execution unit 16 Suspension recovery process execution unit 17 Temperature control unit 21 Cell 22 Suspension

Claims (10)

A passaging assisting device for performing a detachment step when performing a passaging operation on cells respectively accommodated in a plurality of storage units,
a dispensing mechanism for dispensing and dripping the solution;
a container holder that holds a container for culturing cells;
a temperature control mechanism for controlling the temperature of the container;
a culture supernatant removal step execution unit for executing a culture supernatant removal step of removing the medium while leaving the cells from each of the plurality of storage units;
a reagent reaction process executing unit for executing a reagent reaction process of supplying a reagent to each container after the culture supernatant removal step has been executed, and severing the binding of cell adhesion proteins to each container;
a medium supply step execution unit for executing a medium supply step of supplying a medium to each container after the reagent reaction step has been executed;
a medium agitating step execution unit that agitates the medium in each container after the medium supply step is performed to generate a suspension in which cells are mixed in the medium agitating the medium;
a suspension collection step executing unit that executes a suspension collection step of collecting suspension from each storage unit after the medium stirring step is executed,
The temperature control mechanism heats the interior of each container to a temperature at which the cell activity is maintained and the reagent reacts appropriately, at least while the reagent reaction process execution unit is executing the reagent reaction process ,
The culture supernatant removal step, the reagent reaction step, the medium supply step, the medium agitation step, and the suspension collection step are carried out while the container is set in the temperature control mechanism, and A subculture assisting device characterized in that it is carried out . 2. The subculture assisting device according to claim 1, wherein the temperature at which the cell activity is maintained and the reagent reacts appropriately is 33 to 38.degree. At least the medium supply step, the medium agitation step, and the suspension recovery step are performed in a series of steps for each storage unit, so that the series of steps is sequentially performed for each storage unit. The passage assisting device according to claim 1 or 2, characterized by the following. Further comprising a washing process execution unit for executing a washing process of washing the cells by supplying a washing liquid to each container after the culture supernatant removal step is performed and removing the washing liquid after a predetermined time has elapsed,
The reagent reaction step supplies a reagent to each container after the washing step has been performed,
3. The series of steps is sequentially performed for each container by performing at least the culture supernatant removing step and the washing step in a series of steps for each container. 3. The passage assisting device according to 1 or 2. 5. The passage assisting device according to claim 3 or 4, wherein the series of steps for each container is performed sequentially without waiting time for each container. Passaging for performing a detachment step when performing a passaging operation on cells accommodated in a plurality of storage units using the subculture assisting device according to any one of claims 1 to 5 A method of operation comprising:
A culture supernatant removing step of removing the culture medium from each of the plurality of storage units while leaving the cells;
A reagent reaction step of supplying a reagent to each container after the culture supernatant removal step is performed, and removing the reagent after a predetermined time has elapsed, thereby severing the binding of cell adhesion proteins to each container;
a medium supply step of supplying a medium to each container after the reagent reaction step has been performed;
A medium stirring step of generating a suspension in which cells are mixed in the medium by stirring the medium in each container after the medium supplying step is performed;
a suspension recovery step of recovering the suspension from each container after the medium stirring step is performed;
and a temperature control step of heating the inside of each container to a temperature at which the cell activity is maintained and the reagent reacts appropriately at least during the time the reagent reaction step is being performed. Method. 7. The subculture operation method according to claim 6, wherein the temperature at which the cell activity is maintained and the reagent reacts appropriately is 33 to 38°C. At least the medium supply step, the medium agitation step, and the suspension recovery step are performed in a series of steps for each storage unit, so that the series of steps is sequentially performed for each storage unit. The subculture operation method according to claim 6 or 7, characterized by: Further comprising a washing step of washing the cells by supplying a washing solution to each container after the culture supernatant removal step is performed and removing the washing solution after a predetermined time has elapsed,
The reagent reaction step supplies a reagent to each container after the washing step has been performed,
3. The series of steps is sequentially performed for each container by performing at least the culture supernatant removing step and the washing step in a series of steps for each container. The subculture operation method according to 6 or 7. 10. The passaging operation method according to claim 8 or 9, wherein the series of steps for each container is performed sequentially without waiting time for each container.

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