JPH04234979A - Apparatus for culturing cell - Google Patents

Abstract

PURPOSE:To reduce shearing force applied to cultured cells in exchanging a culture solution. CONSTITUTION:The aforementioned apparatus for culturing cells is equipped with a cell culture vessel 1, a gas feeding means for feeding a pressurized gas for controlling a culture solution to the cell culture vessel 1, a cloth filter 22 for separating the old culture solution from the cells and a culture solution conveying means for conveying the culture solution in the cell culture vessel 1 to the cloth filter 22 using a pressure from the gas feeding means.

Description

[Detailed description of the invention]

0001

[Industrial Field of Application] The present invention relates to a cell culture device, particularly,
The present invention relates to a cell culture device for culturing cells by storing cells and a culture solution in a container.

0002

2. Description of the Related Art A conventional cell culture device equipped with a cell culture container for storing cells and a culture solution is known. The device is a separation device (
It has a circulation path equipped with a filter) and a peristaltic pump. In the circulation route, a peristaltic pump is used to extract the culture solution from the cell culture container, and the old culture solution is collected through a separation device. In addition, an amount of new culture solution corresponding to the amount of recovered old culture solution can be supplied into the cell culture container from another route.

0003

SUMMARY OF THE INVENTION The conventional cell culture apparatus described above employs a structure in which a peristaltic pump is used to extract the culture solution from the cell culture container. Since this peristaltic pump has a structure in which liquid is circulated by handling a flexible tube, it has the advantage that it is difficult for bacteria to enter from the outside. However, when culturing animal cells without cell walls or cells that require the use of carrier beads, these cells are susceptible to shearing forces, and the operation of the pump has an adverse effect on the cells.

[0004] An object of the present invention is to provide a cell culture device that can reduce the shearing force applied to cultured cells when exchanging the culture medium.

[0005]

[Means for Solving the Problems] A cell culture device according to the present invention includes a cell culture container for storing cells and a culture solution, and a cell culture container for supplying pressurized gas for controlling the culture solution to the cell culture container. A gas supply means, a separation device for separating old culture solution and cells, a culture solution conveyance means for transferring the culture solution in the cell culture container to the separation device, and a new culture solution to the cell culture container. and a culture solution supply means for supplying the culture solution. In this cell culture apparatus, the culture solution transport means transports the culture solution using pressurizing force from the gas supply means.

[0006]

[Operation] In the cell culture apparatus according to the present invention, cells and a culture solution are stored in a cell culture container. The state of the culture solution in the cell culture container is controlled by the control pressurized gas from the gas supply means.

[0007] When exchanging the culture solution in the cell culture container, the culture solution transfer means uses the pressure from the gas supply means to transfer the culture solution in the cell culture container to the separation device. The separation device separates old culture fluid from cells. A culture solution supply means supplies new culture solution to the cell culture container from which the old culture solution has been removed.

[0008] In this case, since the culture solution transport means uses the pressurizing force from the gas supply means to transport the culture solution to the separation device, there is no need to use a pump that applies shear force to the cultured cells. Shearing force on cultured cells can be reduced when replacing the culture solution.

[0009]

Embodiment FIG. 1 shows a cell culture apparatus as an embodiment of the present invention. This cell culture device includes a culture container 1 containing cells and a culture solution, a container 2 containing a new culture solution, and a container 3 containing an old culture solution.

[0010] The top opening of the culture container 1 is sealed with a lid 4. The lid 4 has a temperature sensor 5 and a pH sensor 6.
and a DO sensor 7 are attached. Further, a liquid level sensor 8 is provided on the side surface of the culture container 1 to detect the amount of culture solution in the container 1. A stirrer 9 for stirring is arranged inside the container 1, and a heater 10 for heating is provided below the container 1.

[0011] The culture vessels 1 and 2 are connected by a conduit 11, and new culture solution can be supplied from the vessel 2 to the culture vessel 1 through the conduit 11. A second pump 12 made of a peristaltic pump is provided in the middle of the conduit 11. Furthermore, one end of a pipe line 13 for supplying pressurized gas for controlling the culture solution is connected to the culture container 1. The other end of the conduit 13 branches into two. An N2 cylinder, an O2 cylinder, and a CO2 cylinder (not shown) are connected to one branch path 14 via an air filter 15 and a solenoid valve 16, respectively.

The other branch 15 of the pipe 13 and the culture container 1
A culture solution exchange path 20 is provided between the two. The exchange path 20 is provided with a liquid reservoir 21 and a cross filter 22 in this order from the branch path 15 side. Cross filter 22
is for separating cells from culture medium. A conduit 23 is provided between the container 3 and the outlet 22a of the cross filter 22 from which the old culture solution from which cells have been separated is taken out. A first pump 24 made of a peristaltic pump is arranged in the middle of the conduit 23.

Furthermore, the culture container 1 is provided with an exhaust pipe 25.
is connected. The tip of the pipe line 25 is open to the atmosphere, and a check valve 26 and a solenoid valve 27 are provided in the middle.

A first valve 30 consisting of a pinch valve is provided in a portion of the exchange path 20 between the culture container 1 and the cross filter 22 and a portion of the pipe line 25 between the check valve 26 and the solenoid valve 27. attached. In addition, branch roads 14 and 15
A second valve 31 having the same structure as the first valve 30 is attached to the second valve 31 .

Next, the structure of the second valve 31 will be explained. Note that since the first valve 30 has the same structure as the second valve 31, a description thereof will be omitted here.

As shown in FIG. 2, the second valve 31 has a valve body 32 for opening and closing the branch passages 14 and 15, and a drive section 33 for driving the valve body 32. As shown in FIG. 3, the valve body 32 includes a generally cylindrical case 34 and a drive member 35 housed within the case 34.
and a pair of pins 36 fixed in the case 34 in its diametrical direction. Branch paths 14 and 15 are case 3
4 and is sandwiched between a drive member 35 and a pin 36.

The drive unit 33 includes a case 37 to which a case 34 is fixed, and a solenoid 38 disposed inside the case 37.
and a rod 39 within the solenoid 38 and driven by the solenoid 38. A driving member 35 is fixed to the tip of the rod 39. That is, by moving the rod 39 in the axial direction, the driving member 3
5 moves in the axial direction within the case 34.

Furthermore, this cell culture apparatus has a control section 40 as shown in FIG. The control unit 40 is a CPU
, ROM, RAM, etc. The I/O port 41 of the control unit 40 includes the stirrer 9 described above, the heater 10, the sensors 5, 6, 7, 8, the first valve 30, the second valve 31, and the first pump 2.
4, the second pump 12, and solenoid valves 16 and 27 are connected. Further, the I/O port 41 is connected to an input panel 42 through which an operator inputs commands and other input/output units.

Next, the operation of the above embodiment will be explained with reference to the control flowcharts shown in FIGS. 5 and 6. When the program starts, initial settings such as checking the operation of various sensors are performed in step S1 in FIG. Once the initial settings have been completed, an operation start command from the input panel 42 is awaited in step S2. After the operator performs setting operations such as putting the culture solution and cells into the culture container 1, the operator issues an operation start command through the input panel 42. Thereby, the program moves from step S2 to step S3.

In step S3, the valves 30 and 31 are driven to open the pipe line 25 and branch line 14, and close the exchange line 20 and branch line 15. That is, in FIG. 1, the first valve 30 is opened on the ■ side, and the second valve 31 is opened on the ■ side. This simultaneously closes the first valve 30 on the ■ side and the second valve 31 on the ■ side.

[0021] In step S4, the stirrer 9 is driven,
Start stirring. In step S5, based on the detection result from the temperature sensor 5, the culture vessel 1 is heated using the heater 10.
Controls the internal temperature. In step S6, the pH sensor 6
pH control is performed based on the detection results. Furthermore, in step S7, DO control is performed based on the detection result from the DO sensor 7. Control in step S6 and step S7 is performed by opening and closing the solenoid valve 16 to control N2 gas, O2 gas,
This is done by supplying gas or CO2 gas to the culture vessel 1.

[0022] In step S8, it is determined whether it is time to replace the culture medium. This determination is made based on determining whether a predetermined time has elapsed since the start of culture (step S2) or whether a predetermined time has elapsed since the previous culture medium exchange. If the predetermined time has not elapsed, the program moves from step S8 to step S9. In step S9, it is determined whether the operator has input a command to terminate the culturing operation via the input panel 42. If the termination command has not been input, the program returns to step S5 and repeats the following processing. Input panel 42
If a command to end the culturing operation is input from , the program moves from step S9 to step S10, and the stirrer 9
to stop. When the process in step S10 is finished, the process returns to step S2.

During the above-mentioned culture operation, if it is determined that it is time to replace the medium, the program proceeds to step S.
8 to step S11, and the medium exchange subroutine of FIG. 6 is executed. In FIG. 6, in step S13, the first valve 30 is driven in the direction of opening the ■ side. Thereby, the conduit 25 is closed. Next, in step S13, N2 gas is supplied to the culture container 1 by opening the solenoid valve 16. As a result, the inside of culture container 1 is N2
Pressurized by gas. As a result, the culture solution in the culture container 1 is transported to the cross filter 22 side through the exchange path 20. In step S14, the first pump 24 is driven. As a result, the culture solution and cells are separated in the cross filter 22, and the old culture solution is stored in the container 3 through the conduit 23. The culture solution with increased cell density is
The liquid is stored in the liquid reservoir 21.

[0024] When the time required for the pressures in the liquid reservoir 21 and the culture container 1 to become approximately equal has elapsed, step S15
The process then moves to step S16. In step S16,
By closing the solenoid valve 16, the supply of N2 gas to the culture container 1 side is stopped. In step S17, the first pump 24 is stopped. In step S18, the first valve 30 is driven so that the ■ side opens. As a result, the gas inside the culture container 1 is discharged through the conduit 25, and the inside of the culture container 1 returns to atmospheric pressure. When the predetermined time required for the inside of the culture container 1 to return to atmospheric pressure has elapsed, the program returns to step S.
19, the process moves to step S20.

In step S20, the second valve 31 is
Drive so that the side opens. As a result, the culture solution in the liquid reservoir 21 which is in a high pressure state is returned through the pipe line 13 into the culture container 1 which is at atmospheric pressure. When sufficient time has elapsed for the culture solution in the liquid reservoir 21 to return to the culture container 1, the program moves from step S21 to step S22. In step S22, the second valve 31 is driven so that the ■ side opens. In step S23, the second pump 12 is driven. As a result, the new culture solution stored in the container 2 is transported to the culture container 1 through the pipe line 11. Step S
In step 24, it is determined whether the amount of culture solution in the culture container 1 has reached a predetermined value based on the detection result from the liquid level sensor 8. If the amount of culture solution in the culture container 1 reaches a predetermined value, the process moves to step S25. In step S25, the second pump 12 is stopped, and the supply of new culture solution to the culture container 1 is stopped. When the process in step S25 is completed, the program returns to the main routine of FIG.

As explained above, in the above embodiment,
When discharging the old culture solution, the culture solution is supplied from the culture container 1 to the cross filter 22 side using the pressurizing force of the gas, so that strong shearing force is not applied to the cells moving through the exchange path 20. Therefore, even when cells that are sensitive to shearing force are cultured, there is little damage to the cells when the culture solution is replaced.

[0027]

Effects of the Invention In the cell culture apparatus according to the present invention, the culture solution conveying means uses the pressurizing force from the gas supply means to convey the culture solution in the cell culture container to the separation device. Therefore, according to the present invention, it is possible to reduce the shearing force applied to the cultured cells when replacing the culture solution.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of an embodiment of a cell culture device according to the present invention.

FIG. 2 is a perspective partial view of the liquid reservoir portion.

FIG. 3 is a partial perspective cross-sectional view of the pinch valve.

FIG. 4 is a schematic block diagram of the control unit.

FIG. 5 is a control flowchart.

FIG. 6 is a flowchart of the subroutine of FIG. 5;

[Explanation of symbols]

1　Culture container 2,3 Container 12 Pump 16 Solenoid valve 22 Cross filter 30 1st valve 31 Second valve

Claims (1)

[Claims] 1. A cell culture container for storing cells and a culture solution, a gas supply means for supplying pressurized gas for controlling the culture solution to the cell culture container, and a cell culture container for storing old culture solution and cells. a separation device for separating; a culture solution conveying device for conveying the culture solution in the cell culture container to the separation device using pressurizing force from the gas supply device; A cell culture device comprising: a culture solution supply means for supplying a culture solution.