JP2002153256A - System for exchanging culture medium of cell culture container - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compact system for exchanging the culture medium of a cell culture container, which can suitably exchange the culture medium in the cell culture container, can carrying out the substantially same cell culture test as a cell culture test on the ground, and is excellent in workability. SOLUTION: This system for exchanging the culture medium of the cell culture container is characterized by comprising a culture medium-receiving means 22 for storing a fresh culture medium and a waste culture medium- receiving means 23 disposed adjacently to the culture medium-receiving means 22, a culture medium-supplying pipe 25 for connecting the culture medium- receiving means to the cell culture container and a culture medium-discharging pipe 27 for connecting the cell culture container to a waste culture medium- receiving means, connectors 31a, 31b detachably connected to the culture medium-injecting or extracting holes 5, 6, respectively, of the cell culture container disposed at the ends of the culture medium-charging pipe 25 and the culture medium-discharging pipe 27, and a liquid-sending pump 28 for supplying the culture medium in the culture medium-receiving means 22 to the cell culture container at least through the culture medium-supplying pipe 25.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a medium exchange system used for exchanging medium 0 in a cell culture vessel when culturing and testing cells such as animal cells under microgravity. .

[0002]

2. Description of the Related Art Conventionally, when performing a culture test of animal cells under microgravity such as in outer space, the culture test is carried out in a single cell culture vessel in which the medium is full, No. 7-8260, connecting a pipe with a medium circulation pump to a cell culture vessel,
A method of culturing while continuously circulating a medium is known.

[0003] In general, when conducting a culture test of animal cells of this kind on the ground, the animal cells are observed in a stationary state, and the internal medium containing an increased amount of waste components is replaced with a new one as needed. Flask culture is performed, and there is a demand for performing a culture test in the same observation environment in space.

[0004]

However, depending on the method of circulating the culture medium in the cell culture vessel, it is not possible to observe the cell culture in a stationary state as described above because the culture medium is constantly flowing. . In addition, in the method of performing a culture test using a single cell culture container, when the medium needs to be changed, the medium must be changed manually, which is inferior in workability. Was.

[0005] The present invention has been made in view of the above circumstances, it is possible to replace the medium in the cell culture container appropriately,
An object of the present invention is to provide a compact and excellent workability medium exchange system for a cell culture container, which can perform a cell culture test substantially similar to flask culture on the ground.

[0006]

According to a first aspect of the present invention, there is provided a system for exchanging culture medium for a cell culture container according to the present invention, comprising a culture medium storage means for storing fresh culture medium and a waste medium provided adjacent to the culture medium storage means. Medium storage means, a medium supply pipe connecting the medium storage means and the cell culture container and a medium discharge pipe connecting the cell culture container and the waste medium storage means, and an end portion of the medium supply pipe and the medium discharge pipe. A connector provided to be detachably connected to a culture medium injection hole and a culture medium discharge hole of a cell culture container, and a liquid supply interposed at least in a culture medium supply pipe to supply the culture medium in the culture medium storage means to the cell culture container. And a pump.

According to a second aspect of the present invention, the liquid feed pump according to the first aspect is a peristaltic pump having two sets of a suction port and a discharge port, and one of the suction port and the discharge port. Is connected to the culture medium supply pipe, and the culture medium discharge pipe is connected to the other suction port and discharge port.

[0008] The invention described in claim 3 is the first invention.
Or In the invention according to 2, wherein the cell culture vessel is
An oxygen supply means for supplying oxygen to the space is provided in a plate-shaped main body in which a space that penetrates between both surfaces and is filled with a medium is provided, and the medium supply pipe is provided in the main body. The injection hole that is connected to inject the medium into the space, and the pouring hole that is connected to the medium discharge pipe and discharges the medium from the space is provided. Is what you do. Here, various forms can be applied as the cell culture container. For example, a plate-shaped main body in which a space penetrating between both surfaces and filled with a medium is formed, and one of the main body is A culture plate having a cell-adhesive surface on the space portion side, which is disposed on the surface and covers the space portion, and a transparent and oxygen-permeable surface disposed on the other surface of the main body and covering the space portion. It is preferable that the membrane material as the oxygen supply means is detachably integrated in a liquid-tight manner.

According to a fourth aspect of the present invention, in the third aspect of the present invention, the injection hole and the injection hole of the cell culture vessel each have one end opening at the opposite end of the space. The other end is drilled so as to open adjacent to the same side surface of the main body, and the connection ends of the culture medium supply pipe and the culture medium discharge pipe are simultaneously connected to each other by one operation. A connector for connecting the portion to the injection hole and the discharge hole. Further, the invention described in claim 5 is the invention according to claims 1-4.
The medium supply pipe according to any one of the above, wherein a gas separation means is interposed.

In the invention according to any one of the first to fifth aspects, the connectors of the medium supply pipe and the medium discharge pipe are connected to the medium injection hole and the discharge hole of the cell culture vessel, respectively, and the liquid supply pump is operated. Then, the fresh medium in the medium storage means is supplied from the medium supply pipe into the cell culture vessel, and the old medium in the cell culture vessel is discharged from the medium discharge pipe to the waste medium storage means in one direction. The medium exchange is performed by forming the flow of the medium.

At this time, the medium can be exchanged by one operation of operating the liquid feeding pump, so that the workability is excellent and the medium storing means and the waste medium storing means are disposed adjacent to each other. Therefore, a large shift of the center of gravity does not occur due to the medium exchange. In addition, connectors are provided at the ends of the medium supply pipe and the medium discharge pipe, which are detachably connected to the medium injection hole and the medium discharge hole of the cell culture vessel, respectively. Prepare a cell culture vessel with cells attached,
By launching this into space, carrying it into the medium exchange system installed in an experimental facility such as a spacecraft, and connecting it to the connector, it becomes possible to perform a culture test on various cells.

According to the second aspect of the present invention, a peristaltic pump having two sets of suction ports and discharge ports is used as the liquid sending pump, and the medium supply pipe is provided at one of the suction port and the discharge port. Is connected, and the culture medium discharge pipe is connected to the other suction port and discharge port, so that the above-described one-way culture medium flow can be formed smoothly.

When conducting a culture test of animal cells, it is necessary to supply oxygen to the animal cells by any method. Since liquid does not substantially separate into two layers, it is not possible to perform a test by stationary observation in a state where gas and liquid are separated into two layers as on the ground. Therefore, as the cell culture vessel used in the present invention, it is preferable that the main body of the cell culture vessel is provided with an oxygen supply means such as a membrane material for supplying oxygen to the space, as in the invention described in claim 3. It is suitable.

According to the above-mentioned cell culture vessel, oxygen is always supplied to the space where cells are cultured by the oxygen supply means. Therefore, as in the system disclosed in Japanese Patent Application Laid-Open No. 7-8260, oxygen is separately supplied. No additional equipment is required, and the accompanying equipment is simplified. Further, for example, a culture plate having a cell-adhesive surface is provided on one surface of the above-described main body, and a transparent and oxygen-permeable membrane material covering the space is provided as an oxygen supply means on the other surface. If the thing is used, on the ground or the like, the cells are attached to the surface of the culture plate in advance, and after integrating this with the main body and the membrane material, it is possible to supply the medium inside,
By observing the cells in the space from the transparent membrane material after supplying the culture medium, it is possible to easily perform a stationary test in a liquid-full state simulating a flask on the ground.

Furthermore, in the invention according to claim 4, one end of the injection hole and the one end of the injection hole in the cell culture vessel are formed so as to open to the opposite ends of the space. By injecting a new culture medium through the injection hole that opens in one of the spaces, the old culture medium can be smoothly discharged from the pouring hole that opens in the other of the space. In addition, the connection ends of the injection hole and the discharge hole are formed so as to open on the same side surface of the main body, and a connector that can be connected to both of the connection ends simultaneously by one operation as a connector to be connected thereto. Is used, the work of connecting the culture medium supply pipe and the culture medium discharge pipe to the injection hole and the discharge hole when exchanging the culture medium becomes easy even in a small test room in outer space.

In the invention according to any one of the first to fourth aspects, since the medium exchange is performed smoothly in one direction of the flow of the medium as described above, the airtightness of the cell culture container, the connector, etc. As long as the property is maintained, there is no danger of gas being mixed into the system, but if the gas supply means is interposed in the medium supply pipe as in the invention according to claim 5, the system should be used. Even when gas is mixed in the gas, the gas can be removed by the gas separating means, and the stability of the system can be secured.

[0017]

1 to 3 show one embodiment of a system for exchanging a culture medium of a cell culture vessel according to the present invention. First, the configuration of a cell culture container suitably used in this medium exchange system will be described with reference to FIGS. 2 and 3. Reference numeral 1 in the figure denotes a predetermined strength and corrosion resistance of a fluororesin, polycarbonate, acetal, or the like. It is a main body formed in a rectangular plate shape by synthetic resin.

A hole penetrating between the upper and lower surfaces is formed in the center of the main body 1, and a space 2 in which the medium is filled is formed in the hole. Here, the hole has a ridge line that is parallel to each other at a central portion in the longitudinal direction, and is inclined in a V-shape at both ends.
It is formed in a square shape. Furthermore, in this ridgeline part,
Steps 3a and 3b are formed on the entire upper and lower surfaces, respectively, and grooves 4a and 3b are formed on the outer peripheral side of the steps 3a and 3b.
4b is formed.

The main body 1 has one end opening at one end 2a in the longitudinal direction of the space 2 and the other end opening at the side 1a of the main body. Other end 2
b, an injection / pouring hole 6 extending along the side of the main body 1 and also opening on the side surface 1a of the main body. In addition, in the opening portion on the side surface 1a of the injection / pouring hole 5, 6, a connector 5a for connecting a culture medium supply pipe 25 and a culture medium discharge pipe 27, which will be described later, when performing culture medium exchange as necessary. 6a is fitted, and the side surface 1a
Is provided with a metal mounting plate 19 via a screw 18. Note that, in the present embodiment, the injection / pouring hole 6 is formed in a groove shape that opens on the upper surface of the main body 1 so that the main body 1 can be easily manufactured. It is closed by a gasket 7 made of silicon rubber having the same planar shape as the main body 1 provided.

A culture plate 9 is provided on a step 3b formed on the lower surface side of the main body 1 via a gasket 8 made of silicon rubber fitted into the groove 4b. The culture plate 9 has a surface formed in a plate shape from a material such as polystyrene having cell adhesion, and is slightly smaller than the space portion 2 so as to engage with the step portion 3b and close the space portion 2. It is formed in a large similar hexagonal shape. Further, on the lower surface side (outer surface side) of the culture plate 9, a lower support plate 10 is disposed.

The lower support plate 10 is a plate-like member whose outer shape is made of titanium, stainless steel, aluminum or the like and has the same dimensions as the main body 1. Are formed. In this opening, a step 10a having substantially the same size as the culture plate 9 is formed, and the outer periphery of the culture plate 9 is supported by the step 10a. On the other hand, on the upper surface side of the main body 1, a film material 12 is provided via a gasket 11 made of silicon, viton, or the like fitted into the groove 4a of the step portion 3a. The thickness of the main body 1 is set so that the distance between the culture plate 9 and the membrane 12 is 5 mm or less.

The film material 12 is transparent, for example, made of fluororesin, silicone, polymethylpentene, etc.
It is a thin-walled material having oxygen permeability and is formed so as to have the same hexagonal shape as the culture plate 9, and is disposed so as to cover the entire surface of the space 2. And in the step part 3a,
For supporting the outer peripheral portion of the film material 12 with the
A holding plate 13 formed of a hexagonal frame made of polycarbonate or the like is provided, and an upper support plate 14 is provided on an upper surface of the holding plate 13. The upper support plate 14 is a plate-like member having the same outer shape as the main body 1 made of titanium, stainless steel, aluminum, or the like, similar to the lower support plate 10. An opening 14a having the same shape is formed.

Then, the membrane material 12, the main body 1 and the culture plate 9 arranged as described above are sandwiched between the upper support plate 14 and the lower support plate 10, and are inserted into the square corners of each other. The screw 15 is integrated so as to be detachable. As a result, the culture plate 9 and the membrane 1
2 are disposed so as to be located inside the screw 15 without the screw 15 being inserted at the corners. Further, concave portions 16 are formed at corresponding positions on both sides of the upper surface of the upper support plate 14 and the lower surface of the lower support plate 10 in the longitudinal direction, and the clip 17 is elastically fitted between the concave portions 16. Thereby, higher liquid tightness in the space 2 can be obtained.

FIG. 1 shows a system according to the present embodiment for exchanging the culture medium of the cell culture container having the above-described configuration. This system is provided in a rack 21 incorporated in a canister 20 having a gas permeable filter 20a. Pairs (two upper and lower pairs in the figure) are attached. In FIG. 1, reference numeral 22 denotes a medium storage container (medium storage means) filled with fresh medium, and a waste medium storage container (waste medium storage means) 23 is provided adjacent to the medium storage container 22. I have. A medium supply tube 25 is detachably connected to the medium storage container 22 via a connector 24, and a medium discharge tube 27 is detachably connected to the waste medium storage container 23 via a connector 26. .

The medium supply pipe 25 and the medium discharge pipe 27 are provided with a liquid supply pump 28. The liquid feed pump 28 is a peristaltic pump having two sets of suction ports 29a and 29b and discharge ports 30a and 30b. The medium supply pipe 25 is connected to one of the suction ports 29a and the discharge port 30a, and the other is connected to the other. The culture medium discharge pipe 27 is connected to the suction port 29b and the discharge port 30b.

Further, the fittings 5a, 6a provided in the injection / injection holes 5, 6 of the cell culture container are simultaneously connected to the tips of the medium supply pipe 25 and the medium discharge pipe 27 by one operation. Connectors 31a and 31b that can be detachably connected to the respective connecting tools 5a and 6a are attached. Further, a gas separator (gas separation means) 32 composed of a hydrophobic film and a hydrophilic film is provided in the medium supply pipe 25 for removing the gas when the gas enters the system. ing. The gas separator 32
Can be omitted. In addition, the code | symbol 33 in a figure is
The properties of the medium in the cell culture vessel are detected,
Is a circuit board in which a control circuit for automatically exchanging the culture medium in the space 2 by starting and stopping is installed.

According to the system for exchanging the culture medium of the cell culture container having the above configuration, the cell culture container alone or the connection tool 5a of the injection / injection hole 5, 6 of the cell culture container,
With the connectors 31a and 31b connected to 6a, respectively, the culture state of the animal cells A attached to the culture plate 9 from the transparent membrane material 12 side can be statically observed. At this time, the membrane 12 has oxygen permeability and is set so that the distance between the culture plate 9 and the membrane 12 is 5 mm or less. 2
The cell A attached to the culture plate 9 in the inside can always be supplied with uniform oxygen. As a result, since a separate facility for supplying oxygen is not required as in the case of the conventional container, the accompanying facilities can be simplified.

When a certain period of time elapses and wastes increase in the culture medium in the space 2, a signal for driving the liquid feed pump 28 is sent from the circuit board 33 based on the detection signal and the waste signal is sent. The liquid feed pump 28 operates. As a result, the fresh culture medium in the culture medium storage container 22 is sent from the culture medium supply pipe 25 to the injection / injection hole 6, and from one end 2b of the space 2 to the space 2
And the old culture medium in the space 2
From the medium discharge pipe 27 to the waste medium storage container 23 through the injection / injection hole 5.

As described above, according to the system for exchanging the culture medium of the cell culture container having the above configuration, the medium supply tube 25 and the medium discharge tube 27 are connected to the medium injection / injection holes 5 and 6 of the cell culture container, respectively. When the liquid supply pump 28 is operated by connecting the base plates 31a and 31b, the fresh medium in the medium storage container 22 is supplied from the medium supply tube 25 into the cell culture container, and the old medium in the cell culture container is replaced with the medium discharge tube. Since the one-way flow of the medium discharged from 27 to the waste medium storage container 23 is formed and the medium is exchanged, the medium exchange can be smoothly performed by one operation of operating the liquid feed pump 28. It is possible to perform the operation, and thus the workability is excellent. In addition, since the medium storage container 22 and the waste medium storage container 23 are disposed adjacent to each other, a large movement of the center of gravity occurs due to the exchange of the medium. That there is no.

Further, a peristaltic pump having two sets of suction ports 29a, 29b and discharge ports 30a, 30b is used as the liquid sending pump 28, and the medium supply pipe 25 is connected to one of the suction port 29a and the discharge port 30a. Since the culture medium discharge pipe 27 is connected to the other suction port 29b and the discharge port 30b, the flow of the above-described one-way culture medium can be smoothly formed.

The medium supply pipe 25 and the medium discharge pipe 2
7 are provided with connectors 31a and 31b which are detachably connected to the medium injection / injection holes 5 and 6 of the cell culture container, so that the cells can be attached to the surface of the culture plate 19 on the ground or the like. The cell culture vessel prepared above is prepared, launched into space, carried into the above-mentioned medium exchange system installed in an experimental facility such as a spacecraft, and connected to the connectors 31a and 31b.
, It becomes possible to conduct a culture test on various cells.

At this time, the connector 5 for the injection / ejection holes 5, 6
a, 6a are formed so as to open on the same side surface 1a of the main body 1, and as the connectors 31a, 31b to be connected to them, the respective connecting tools 5a, 6a are simultaneously operated by one operation
Since it uses a device that can be connected to the medium, it can be used for injection and
The work of connecting the culture medium supply pipe 25 and the culture medium discharge pipe 27 to the pouring holes 5, 6 can be easily performed.

[0033]

As described above, according to the system for exchanging the culture medium of the cell culture container according to any one of claims 1 to 5, the connector is connected to the cell culture container and the liquid supply pump is operated. Thereby, a one-way flow of the medium is formed, and the medium can be exchanged in the cell culture container, so that the workability is excellent, and the medium storing means and the waste medium storing means are disposed adjacent to each other. Therefore, a large shift of the center of gravity does not occur due to the medium exchange. Moreover,
A cell culture container having cells attached to the surface of a culture plate is prepared on the ground or the like by the connector, launched into space, and transported to the medium exchange system installed in an experimental facility such as a spacecraft. By connecting to the connector, a culture test for various cells can be performed.

According to the second aspect of the present invention, a peristaltic pump having two sets of suction ports and discharge ports is used as a liquid sending pump, and the medium supply pipe is connected to one of the suction ports and the discharge port. According to the invention as set forth in claim 3, since the connection is made and the culture medium discharge pipe is connected to the other suction port and the other discharge port, the flow of the culture medium in one direction can be formed smoothly. Since oxygen is always supplied to the space for culturing cells by the oxygen supply means, no additional equipment for supplying oxygen is required, and simplification of incidental equipment is achieved.

Further, according to the invention described in claim 4,
One connection operation by a connector allows simultaneous connection to the connection ends of both cell culture vessels, so that even in a small test room in outer space, when exchanging the culture medium, the medium is supplied to the injection hole and the ejection hole. The operation of connecting the tube and the medium discharge tube is facilitated, and according to the invention described in claim 5, even if gas enters the system, the gas can be removed by the gas separating means. Thus, the effect that the stability of the system can be ensured can be obtained.

[Brief description of the drawings]

FIG. 1 shows an embodiment of a system for exchanging a culture medium of a cell culture container according to the present invention.

FIG. 2 is a cross-sectional view showing a cell culture container used in the medium exchange system of FIG.

FIG. 3 is an exploded perspective view of FIG. 2;

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Main body 1a One side of main body 2a, 2b One end of space part 5, 6 Injection / pouring hole 9 Culture plate 12 Membrane material 22 Medium storage container (medium storage device) 23 Waste medium storage container (waste medium storage device) 24, 26, 31a, 31b Connector 25 Medium supply pipe 27 Medium discharge pipe 28 Liquid feed pump 29a, 29b Intake port 30a, 30b Discharge port 32 Gas separator (gas separation means)

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Shigeyuki Ogawa 2-1-1, Tsurumichuo, Tsurumi-ku, Yokohama-shi, Kanagawa Prefecture Inside Chiyoda Kako Construction Co., Ltd. 1-chome No. 1 F-term in Chiyoda Kako Construction Co., Ltd. 4B029 AA02 BB11 CC01 CC02 DA04 DB16

Claims (5)

[Claims] 1. A medium storing means for storing a fresh medium, a waste medium storing means provided adjacent to the medium storing means, a medium supply pipe connecting the medium storing means and a cell culture container, and the cell culture. A medium discharge tube for connecting the container and the waste medium storage means, and detachably connected to the medium injection hole and the medium discharge hole of the cell culture container provided at the ends of the medium supply tube and the medium discharge tube, respectively. And a liquid supply pump interposed at least in the culture medium supply pipe and supplying a culture medium in the culture medium storage means to the culture cell container. system. 2. The liquid supply pump is a peristaltic pump having two sets of suction ports and discharge ports, and the culture medium supply pipe is connected to one of the suction ports and the discharge port.
The system for exchanging culture medium for a cell culture container according to claim 1, wherein the culture medium discharge pipe is connected to the other of the suction port and the discharge port. 3. The cell culture vessel has a plate-shaped main body in which a space that penetrates between both surfaces and is filled with a medium is provided with an oxygen supply unit that supplies oxygen to the space. And the main body is connected to the culture medium supply pipe to inject the culture medium into the space, and the injection hole is connected to the culture medium discharge pipe to discharge the culture medium from the space. The system for exchanging culture medium for a cell culture container according to claim 1 or 2, wherein 4. The injection hole and the pouring hole of the cell culture container each have one end opened at the opposite end of the space, and the other end opened adjacent to the same side surface of the main body. At the connection end of the culture medium supply pipe and the culture medium discharge pipe, a connector for connecting both the connection ends to the injection hole and the discharge hole at the same time by one operation is provided. The system for exchanging culture medium of a cell culture container according to claim 3, wherein 5. The system for exchanging culture medium for a cell culture container according to claim 1, wherein gas separation means is interposed in the culture medium supply pipe.