Cell culture device

Abstract

To provide a method to generate hypoxia without using nitrogen gas in a device for culturing cells.SOLUTION: A culture chamber 3 having heating means to grow cells is a device comprising a reservoir tank 2 with an openable / closable lid, in which a deoxidizer 1 is introduced into the reservoir tank; the culture chamber and the reservoir tank have two inlet / outlet ports for the gas therein respectively; on-off valves 4 and 5 electrically opened and closed are disposed on the inlet / outlet ports of the reservoir tank; the on-off valves are connected to the inlet / outlet ports of the culture chamber by gas tubes 8 and 9; and a pump 11 for circulating gas is disposed in the middle of either of the tubes. One of the two on-off valves 4 and 5 disposed on the cell culture device is a three-way valve 4; a T-shaped joint 12 is disposed in the middle of the tube 8 connecting the other on-off valve 5 and the culture chamber; and the three-way valve is connected in a direction to open / close the gas from the tube on the culture chamber side by selecting either the reservoir tank or the T-shaped joint.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an apparatus for culturing cells.

Background Art

[0002] Patent Document 1 discloses an apparatus for culturing cells in the medical and research fields such as life science and regenerative medicine. It has an environment approximated to that inside the body by controlling temperature, carbon dioxide, and oxygen at a constant level.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

[0004] The temperature can be relatively easily controlled by using a heat source to keep it at a value approximated to body temperature. Carbon dioxide is generally maintained at 5%, but since it is almost 0% in the standard atmosphere, it can be achieved by mixing the carbon dioxide gas filled in a cylinder with the atmosphere. On the other hand, oxygen is 20.9% in the standard atmosphere, but the oxygen concentration is low inside the body. Therefore, to create a similar low-oxygen state, a method is adopted in which a chamber partitioned in a closed space with cells installed inside is filled with nitrogen gas to expel oxygen outside the chamber to create a low-oxygen state.

Disclosure of the Invention

Problems to be Solved by the Invention

[0005] However, in the conventional method, it is necessary to constantly flow nitrogen gas to reduce the oxygen concentration, resulting in rapid consumption and high running costs. Nitrogen gas is filled in a high-pressure cylinder, but since a large amount is required, it becomes a large cylinder, and the installation and management of the cylinder are troublesome.

[0006] In addition, there is a deoxidizer that utilizes the chemical property of adsorbing oxygen when iron oxidizes. There is a method of putting the deoxidizer in a bag to reduce the internal oxygen concentration. However, since the bag is manually opened and closed and mixed with the atmosphere to adjust the concentration, it has been difficult to adjust precisely and for a long period of time.

[0007] The present invention solves these problems, and its object is to provide a culture device that can adjust the oxygen concentration in a small and simple manner.

Means for Solving the Problems

[0008] To achieve the above object, the present device is a cell culture device including a reservoir tank for sealing a deoxidizer and a culture chamber for growing cells. At two entrances and exits for gas to enter and exit provided in the reservoir tank, one three-way valve and one on-off valve are provided respectively, and tubes are provided for each to connect to the gas entrance and exit of the culture chamber. A circulation pump for flowing gas into the culture chamber is provided in the middle of the tube connecting the on-off valve and the culture chamber. A T-joint for branching the tube is provided between the circulation pump and the on-off valve. The three-way valve is arranged in a direction to select and open or close the flow from the culture chamber to either the reservoir tank or the T-joint. The cell culture device is further provided with two T-joints for branching gas in the middle of the tube connecting the culture chamber and the three-way valve, and on-off valves are provided at the ends of each. One of the on-off valves communicates with the atmosphere, and a cylinder filled with carbon dioxide is connected to the other on-off valve. An oxygen sensor and a carbon dioxide sensor for detecting the gas concentration are provided in the middle of one of the tubes. The on-off valves and the three-way valve are operated so that the concentrations detected by each sensor become preset values.

Effects of the Invention

[0009] By making the inside of the reservoir tank in a low-oxygen state, mixing air, and sending the gas that has been reduced to a predetermined oxygen state to the culture chamber, it becomes possible to perform low-oxygen culture inexpensively and simply without using nitrogen gas. Also, by closing the reservoir tank and short-circuiting the gas flow, it becomes possible to adjust the concentration of only carbon dioxide in a space independent of oxygen control, and it is easy to individually adjust the two types of gas concentrations. Furthermore, even if low-oxygen gas is released into the atmosphere when the lid of the culture chamber is opened for operations such as changing the culture solution, and the oxygen concentration temporarily increases, since low-oxygen gas is waiting in the reservoir tank, when the culture chamber is closed again and restarted, it can quickly return to the predetermined concentration.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

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Figure 6

Figure 7

Embodiments for Carrying Out the Invention

[0011] Hereinafter, the best mode in the present invention is shown. (A) Provide two gas inlets and outlets in the reservoir tank 2 where the lid opens and closes, and install a three-way valve 4 and an on-off valve 5 that open and close with electrical signals respectively. The culture chamber 3 is connected to the on-off valve 5 by a tube 8 and to the three-way valve 4 by a tube 9. A circulation pump 11 is installed in the middle of the tube 8 in the direction of sending gas from the reservoir tank 2 to the culture chamber 3. (B) Install a T-joint 12 between the circulation pump 11 and the on-off valve 5. The three-way valve 4 and the T-joint 12 are connected via a tube 10. The three-way valve 4 is connected in a direction that selectively opens and closes the gas flowing from the culture chamber 3 to either the T-joint 12 or the reservoir tank. (C) Install T-joints 13 and 14 in the middle of the tube 9, and install on-off valves 6 and 7 respectively. Connect a cylinder 17 filled with carbon dioxide to the on-off valve 6, and connect the on-off valve 7 to communicate with the atmosphere. (D) Install an oxygen sensor 15 and a carbon dioxide sensor 16 in the tube 9, and install a controller 18 that receives the electrical signal from the sensors and operates the three-way valve 4 and the on-off valves 5 - 7. The present invention has the above structure, and when in use, it is as follows.

[0012] Put the deoxidizer 1 into the reservoir tank 2 and close it. Install a general-purpose container seeded with cells in the culture chamber 3 and close the lid. Immediately, the inside of the reservoir tank becomes a low-oxygen state, but initially, as shown in Fig. 1, the on-off valves 5, 6, and 7 are closed and blocked from the cylinder 17 and the atmosphere, and the three-way valve 4 is open in the direction of the T-joint 12 and the reservoir tank is also in a closed state.

[0013] When the power is turned on, as shown in Fig. 2, the on-off valve 5 opens and the three-way valve 4 opens in the direction of the reservoir tank. The gas that has become a low-oxygen atmosphere due to the oxidation reaction of the deoxidizer inside the reservoir tank 2 is sent to the culture chamber by the circulation pump 11 and circulates between the reservoir tank 2. Since heat is also generated by the oxidation reaction of the deoxidizer 1, it is advisable to use heat-resistant materials for the reservoir tank 2.

[0014] At the same time, the on-off valve 6 is also opened, and the carbon dioxide accumulated in the cylinder 17 flows into the tube 9 to increase the concentration, and circulates between the culture chamber 3 through the reservoir tank 2 by the circulation pump 11.

[0015] As shown in FIG. 6, the controller 18a controls the opening and closing of the three-way valve 4 and the on-off valve 5 according to the concentration detected by the oxygen sensor 15, and the controller 18b controls the on-off valve 6 according to the concentration detected by the carbon dioxide sensor 16. The on-off valve 7 is opened in common by both the controllers 18a and 18b, but the above control may also be covered by the controller 18. The oxygen sensor 15 and the carbon dioxide sensor 16 are provided in the tube immediately after the gas exits from the culture chamber 3 to ensure that the detected concentration value is approximated to the gas concentration in the culture chamber 3.

[0016] When the oxygen concentration detected by the oxygen sensor 15 reaches the set value, as shown in FIG. 3, the controller 18a closes the on-off valve 5 and the three-way valve 4 opens in the direction of the T-joint 12 and short-circuits to the circulation pump 11, and the low-oxygen gas inside the reservoir tank 2 does not flow in any more to maintain the concentration. At this time, by continuously opening the on-off valve 13, only the concentration of carbon dioxide can be increased.

[0017] Since the reservoir tank 2 does not have a pressurizing function, the low-oxygen gas generated by the chemical reaction of the deoxidizer 1 is allowed to flow into the culture chamber by the circulation pump 11. However, when the inflow is stopped to maintain the concentration, if either the inlet or the outlet is closed and the other is open, the low-oxygen gas will gradually flow out of the reservoir tank 2 and the oxygen concentration in the culture chamber 3 cannot be maintained. On the other hand, if the circulation pump 11 is stopped, the circulation for adjusting the concentration of carbon dioxide will also stop. By providing the three-way valve 4 and the on-off valve 5, the low-oxygen gas in the reservoir tank can be blocked and short-circuited from the gas circulation, and it is possible to maintain oxygen at a low concentration with a small and simple structure and adjust the concentration of carbon dioxide. The on-off valve 5 may use a check valve that only flows in the direction from the reservoir tank 2 to the culture chamber 2.

[0018] When the carbon dioxide concentration detected by the carbon dioxide sensor 16 reaches the set value, the controller 18b closes the on-off valve 6 as shown in Fig. 4 to prevent further inflow of carbon dioxide from the cylinder 17 and maintain the concentration. At this time, by opening the on-off valve 5 and opening the three-way valve 4 to the reservoir tank 2, it is possible to reduce only the oxygen concentration.

[0019] When the concentration of the mixed gas reaches the set value for both oxygen and carbon dioxide, the three-way valve 4 and the on-off valves 5 to 7 stop operating and return to the initial state shown in Fig. 1. The three-way valve 4 opens in the direction of the T-joint 12 and short-circuits to the circulation pump 11, and the on-off valve 5 closes to close the reservoir tank 2 so that the low-oxygen gas does not flow into the tube. At the same time, the on-off valve 13 closes to prevent the release of carbon dioxide, and the on-off valve 14 closes to cut off the atmosphere.

[0020] In Fig. 1, the culture chamber 3 becomes a closed space completely independent of the inflow of gas or the atmosphere, and the two types of gases are mixed by circulating to maintain a concentration suitable for culturing. At this time, the circulation pump may continue to circulate, but by stopping it, gas leakage can be minimized. Or by intermittently repeating stop and circulation, gas leakage can be minimized and diffusion can be performed periodically to equalize the concentration.

[0021] Gas may flow in excessively due to the delayed response of the operation of the three-way valve and the on-off valve, causing the concentration to deviate from the set value. When the oxygen concentration drops too much or the carbon dioxide concentration rises too much as shown in Fig. 5, the controller 18a, 18b, or 18 which combines both opens the on-off valve 14 to communicate with the atmosphere and exhaust the internal gas to adjust the concentration.

[0022] At this time, both oxygen and carbon dioxide gases are exhausted. If one returns to the set value while the other exceeds it, the concentration is adjusted individually as shown in Fig. 3 or Fig. 4.

[0023] Also, as time passes, gas leaks slightly and the concentration becomes insufficient. In that case, the concentrations of oxygen and carbon dioxide are adjusted in parallel as shown in the flowchart of Fig. 7, such as from

[0013] to

[0021] .

[0024] A check valve is provided in the culture chamber 2 and connected to the tube, so that the gas inside can be retained even when the tube is removed, and it can also be temporarily used for transportation or movement.

[0025] Regarding the temperature, it is kept warm by the heat source 19 provided in the culture chamber 2. The heat source may be separately attached with a temperature sensor and maintained at the set temperature by a regulator for the heat source.

Description of Symbols

[0026] 1 Oxygen absorber 2 Reservoir tank 3 Culture chamber 4 Three-way valve 5, 6, 7 On-off valve 8, 9, 10 Tube 11 Circulation pump 12, 13, 14 T-joint 15 Oxygen sensor 16 Carbon dioxide sensor 17 Cylinder 18 Controller 18a Controller 1 18b Controller 2 19 Heat source

Claims

【Claim 1】 A culture chamber having a heating means for culturing cells, a reservoir tank with an openable and closable lid, and comprising, an oxygen scavenger is introduced into the reservoir tank, two inlets and outlets for internal gas are provided in each of the culture chamber and the reservoir tank, two on-off valves that operate electrically are provided at the inlets and outlets of the reservoir tank, the two on-off valves and the inlets and outlets of the culture chamber are connected by a tube for flowing gas, a pump for circulating gas is provided in the middle of the tube, one of the two on-off valves is a three-way valve, a T-joint is provided in the middle of the tube connecting the other on-off valve and the culture chamber, the three-way valve is connected to the T-joint in a direction that opens and closes in a direction for selecting either the reservoir tank or the T-joint for the gas from the culture chamber, two T-joints are provided in the middle of the tube connecting the three-way valve and the culture chamber, an on-off valve connected to the atmosphere is connected to one of the two T-joints, and an on-off valve connected to a cylinder filled with carbon dioxide is connected to the other, an oxygen sensor and a carbon dioxide sensor are provided between the culture chamber and the two T-joints of the tube, control means for receiving the concentrations of oxygen and carbon dioxide detected by the oxygen sensor and the carbon dioxide sensor is provided, the control means operates each on-off valve and the three-way valve so that the concentrations of oxygen and carbon dioxide fall within preset values. A cell culture apparatus characterized by this.

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