JP3572341B2 - Medium exchange system for cell culture vessels - Google Patents

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a medium exchange system used for exchanging medium 0 in a cell culture container when performing a culture test on cells such as animal cells under microgravity.
[0002]
[Prior art]
Conventionally, when performing a culture test of animal cells under microgravity such as in outer space, the culture test is performed in a single cell culture vessel in which the medium is filled, or disclosed in Japanese Patent Application Laid-Open No. 7-8260. A method is known in which a culture medium is continuously circulated by connecting a pipe provided with a medium circulation pump to a cell culture container as described in US Pat.
[0003]
By the way, when conducting a culture test of this kind of animal cells on the ground, generally, while observing the animal cells in a stationary state, a flask culture in which the internal medium with an increased amount of waste components is replaced with a new one as appropriate. There is a demand for conducting a culture test in the same observation environment in space.
[0004]
[Problems to be solved by the invention]
However, depending on the method of circulating the medium in the cell culture container, the cell culture cannot be observed in a stationary state as described above because the medium is constantly flowing.
Also, in the method of performing a culture test using a single cell culture container, when the medium needs to be replaced, the medium must be replaced manually, which is inferior in workability. Was.
[0005]
The present invention has been made in view of the above circumstances, and allows the medium in a cell culture container to be appropriately changed, and allows a cell culture test to be performed substantially similar to a flask culture on the ground. It is an object of the present invention to provide a system for exchanging a medium of a cell culture container which is excellent in the above.
[0006]
[Means for Solving the Problems]
The system for exchanging culture medium for a cell culture vessel according to the present invention according to claim 1, has a space filled with a culture medium, and a culture medium injection hole and a culture medium discharge hole that open at opposing positions of the space. a cell culture vessel having a waste medium housing means provided adjacent to the medium storage means and the medium containing means storing fresh medium, medium supply pipe connecting the said medium accommodating means and the cell culture vessel and a medium discharge pipe which connects the said cell culture vessel and the waste media housing means, provided at the end of the medium supply pipe and the medium discharge pipe, each of the injection hole and the Note Deana of the cell culture vessel supplying a connector, each of which is detachably connected, it is interposed in at least the medium supply pipe of the medium in said medium accommodating means to the space of the cell culture vessel connector provided in And it is characterized in by comprising a liquid pump.
[0007]
According to a second aspect of the present invention, there is provided the liquid sending pump according to the first aspect, wherein the liquid sending pump is a peristaltic pump having two sets of an inlet and an outlet. A supply pipe is connected, and the medium discharge pipe is connected to the other suction port and discharge port.
[0008]
The invention of claim 3 is the invention according to claim 1 or 2, the cell culture vessel, the space plate-shaped body that is formed of medium is filled through between both surfaces to become the membrane material as an oxygen supply means for chromatic oxygen permeability covering the space portion is provided, and in the body, injecting the medium to the space is the medium supply pipe connected said An injection hole and the pouring hole, which is connected to the medium discharge pipe and discharges the medium from the space, are formed.
Here, as the cell culture vessel, various forms can be applied. 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 A culture plate having a cell-adhesive surface on the side of the space portion, which is disposed on the surface and covers the space portion, and a transparent and oxygen-permeable surface which is disposed on the other surface of the main body and covers the space portion. It is preferable that the membrane material as the oxygen supply means is detachably integrated in a liquid-tight manner.
[0009]
Further, the invention according to claim 4, in the invention described in claim 3, the injection holes and casting Deana of the cell culture vessel, each one end opening at an end opposing the space portion, and 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 both connection ends by one operation. it is characterized in that the connector for connecting through the connector to the injection hole and the discharge hole.
A fifth aspect of the present invention is characterized in that the medium supply pipe according to any one of the first to fourth aspects is provided with a gas separating means.
[0010]
In the invention according to any one of claims 1 to 5, when the medium supply pipe and the medium discharge pipe are connected to the medium injection hole and the discharge hole of the cell culture container, respectively, and the liquid supply pump is operated, One-way in which fresh medium in the medium storage means is supplied from the medium supply pipe to the space in the cell culture container, and old medium in the space is discharged from the medium discharge pipe to the waste medium storage means. The medium exchange is performed by forming the flow of the medium.
[0011]
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 arranged 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 culture medium supply pipe and the culture medium discharge pipe so as to be detachably connected to the connection means for the culture medium injection hole and the culture medium discharge hole of the cell culture vessel. Prepare a cell culture vessel with cells attached to the surface of it, launch it into outer space, carry it into the medium exchange system installed in an experimental facility such as a spacecraft, and connect it to the connector In addition, it is possible to perform a culture test on various cells.
[0012]
According to the invention described in claim 2, a peristaltic pump having two sets of suction ports and discharge ports is used as the liquid sending pump, and the culture medium supply pipe is connected to one of the suction ports and the discharge port. Since the culture medium discharge pipe is connected to the other suction port and discharge port, the above-described one-way culture medium flow can be smoothly formed.
[0013]
When conducting a culture test of animal cells, it is necessary to supply oxygen to the animal cells by any method. However, as is well known, under microgravity such as in space, gas-liquid Since there is no specific two-layer separation, it is not possible to perform a test based on stationary observation in a state where gas-liquid is separated into two layers as on the ground.
Therefore, as the cell culture vessel used in the present invention, it is preferable to provide an oxygen supply means such as a membrane material for supplying oxygen to the space in the main body of the cell culture vessel as in the invention described in claim 3. It is suitable.
[0014]
According to the above-mentioned cell culture container, oxygen is always supplied to the space for culturing cells by the membrane material as the oxygen supply means. Therefore, as in the system described in JP-A-7-8260, oxygen is separately supplied. There is no need for a supply facility, and the accompanying facilities can be simplified. Further, for example, a culture plate having a cell-adhesive surface is provided on one surface of the main body described above, and a transparent and oxygen-permeable membrane material covering the space is provided as an oxygen supply means on the other surface. If a medium is used, it is possible to attach the cells to the surface of the culture plate in advance on the ground or the like, integrate this with the main body and the membrane material, and then supply the medium to the inside. By observing the cells in the space from the transparent membrane material after the supply, it is possible to easily perform a stationary test in a liquid full state simulating a flask on the ground.
[0015]
Further, in the invention according to claim 4, one end of the injection hole and the one end of the pouring hole in the cell culture container are formed so as to open to the opposite ends of the space, so that By injecting a new culture medium from the injection hole that opens on one side, the old culture medium can be smoothly discharged from the pouring hole that opens on the other side 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 narrow test room in outer space.
[0016]
In the invention according to any one of claims 1 to 4, as described above, since the medium exchange is performed smoothly in one direction of the flow of the medium, the airtightness of the cell culture container, the connector, and the like is maintained. There is no danger of gas being mixed into the system as long as it is performed, but if the gas supply means is interposed in the medium supply pipe as in the invention of claim 5, the gas will be contained in the system. Even in the case where is mixed, the gas can be removed by the gas separating means, and the stability of the system can be secured.
[0017]
BEST MODE FOR CARRYING OUT THE INVENTION
1 to 3 show one embodiment of a system for exchanging a medium of a cell culture container according to the present invention.
First, the configuration of a cell culture container suitably used for this medium exchange system will be described with reference to FIGS. 2 and 3. Reference numeral 1 in the figure indicates 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.
[0018]
A hole penetrating between the upper and lower surfaces is formed at the center of the main body 1, and a space 2 into which the medium is filled is formed in the hole. Here, the hole is formed in a hexagonal shape in which the ridge lines are parallel to each other at the center in the longitudinal direction, and are inclined in a V-shape at both ends. Further, steps 3a and 3b are formed on the ridge line portion over the entire circumference on the upper and lower surfaces, respectively, and grooves 4a and 4b are formed on the outer peripheral side of the steps 3a and 3b.
[0019]
The main body 1 also has an injection / pouring hole 5 having one end opening at one end 2a in the longitudinal direction of the space portion 2 and the other end opening at the side surface 1a of the main body, and one end 2b at the other end of the space portion 2 in the longitudinal direction. And 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 / ejection 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 medium exchange as necessary. 6a is fitted, and a metal mounting plate 19 is provided on the side surface 1a via screws 18. In this embodiment, the injection / injection hole 6 is formed in a groove shape opening 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.
[0020]
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, a lower support plate 10 is arranged on the lower surface side (outer surface side) of the culture plate 9.
[0021]
The lower support plate 10 is a plate-shaped member whose outer shape is made of titanium, stainless steel, aluminum, or the like and has the same dimensions as the main body 1, and an opening having the same shape as the space 2 of the main body 1 is formed at the center. ing. A step 10a having substantially the same size as the culture plate 9 is formed in this opening, 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.
[0022]
The membrane material 12 is a transparent and oxygen-permeable thin-walled material made of, for example, fluororesin, silicone, polymethylpentene, or the like, and is formed in the same hexagonal shape as the culture plate 9 by It is arranged so as to cover the entire surface of the space 2. A pressing plate 13 formed of a hexagonal frame made of polycarbonate or the like for supporting the outer peripheral portion of the film material 12 therebetween is provided on the step portion 3a. Is provided with an upper support plate 14 on the upper surface of the device.
The upper support plate 14 is a plate-like member having the same outer dimensions 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.
[0023]
The membrane material 12, the main body 1, and the culture plate 9 arranged in a stacked manner as described above are sandwiched between the upper support plate 14 and the lower support plate 10, and the screws inserted into the square corners of each other. 15 are integrated so as to be detachable. As a result, the culture plate 9 and the membrane material 12 are disposed so as to be located inside the screws 15 without the screws 15 being inserted at the corners. Further, concave portions 16 are formed at corresponding positions on both sides in the longitudinal direction of the upper surface of the upper support plate 14 and the lower surface of the lower support plate 10, and clips 17 are elastically fitted between the concave portions 16. Thereby, higher liquid tightness in the space portion 2 can be obtained.
[0024]
FIG. 1 shows a system according to the present embodiment for replacing a medium in a cell culture container having the above-described configuration. This system includes a plurality of sets (see FIG. 1) in a rack 21 incorporated in a canister 20 having a gas permeable filter 20a. In this case, two sets are mounted above and below).
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 pipe 25 is detachably connected to the medium storage container 22 via a connector 24, and a medium discharge pipe 27 is detachably connected to the waste medium storage container 23 via a connector 26. .
[0025]
A liquid supply pump 28 is interposed between the medium supply pipe 25 and the medium discharge pipe 27. The liquid supply 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.
[0026]
Further, the tips of the culture medium supply pipe 25 and the culture medium discharge pipe 27 are simultaneously connected to the connection tools 5a and 6a provided in the injection and discharge holes 5 and 6 of the cell culture container by one operation. Connectors 31a and 31b that can be detachably connected to the components 5a and 6a are attached. Further, a gas separator (gas separating 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. Note that the gas separator 32 can be omitted. Reference numeral 33 in the figure denotes a circuit board on which a control circuit for detecting the properties of the culture medium in the cell culture container, starting and stopping the liquid feed pump 28, and automatically changing the culture medium in the space 2 is incorporated. It is.
[0027]
According to the system for exchanging the culture medium of the cell culture container having the above configuration, the connectors 31a and 31b are respectively connected to the cell culture container alone or to the connectors 5a and 6a of the injection / injection holes 5 and 6 of the cell culture container. In a state in which is connected, 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 material 12 has oxygen permeability and is set so that the distance between the culture plate 9 and the membrane material 12 is 5 mm or less. The cell A attached to the culture plate 9 in 2 can always be supplied with uniform oxygen. As a result, since separate equipment for supplying oxygen is not required as in the case of the conventional container, simplification of the auxiliary equipment can be achieved.
[0028]
When a certain period of time elapses and waste products 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 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 / pouring hole 6, flows into the space 2 from one end 2b of the space 2, and the old culture medium in the space 2 It is discharged from the other end 2 a through the injection / injection hole 5 to the waste medium storage container 23 from the medium discharge pipe 27.
[0029]
As described above, according to the system for exchanging the culture medium of the cell culture container having the above configuration, the medium supply / injection holes 5 and 6 of the cell culture container are provided with the connectors 31a and 31b of the culture medium supply tube 25 and the medium discharge tube 27, respectively. Is connected, and the liquid feed pump 28 is operated, 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 discarded from the medium discharge tube 27. Since a one-way flow of the medium discharged to the 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. Since the medium storage container 22 and the waste medium storage container 23 are arranged adjacent to each other, the center of gravity does not largely move due to the exchange of the culture medium.
[0030]
Moreover, a peristaltic pump having two sets of suction ports 29a and 29b and discharge ports 30a and 30b is used as the liquid sending pump 28, and the culture medium supply pipe 25 is connected to one of the suction ports 29a and the discharge port 30a, and the other is connected. Since the culture medium discharge pipe 27 is connected to the suction port 29b and the discharge port 30b, the above-described one-way flow of the culture medium can be formed smoothly.
[0031]
In addition, connectors 31a and 31b are provided at the ends of the medium supply pipe 25 and the medium discharge pipe 27 so as to be detachably connected to the medium injection / injection holes 5 and 6 of the cell culture container. A cell culture vessel having cells adhered to the surface of the culture plate 9 is prepared, launched into space, and carried into the above-mentioned medium exchange system installed in an experimental facility such as a spacecraft. , It becomes possible to conduct a culture test on various cells.
[0032]
At this time, the connecting tools 5a, 6a of the injection / pouring holes 5, 6 are formed so as to open on the same side surface 1a of the main body 1, and are simultaneously connected by one operation as connectors 31a, 31b connected to these. Since a connector that can be connected to each of the connectors 5a and 6a is used, even in a small test room in outer space, when exchanging the culture medium, the medium supply pipe 25 and the medium discharge pipe are inserted into the injection / injection holes 5 and 6. The work of connecting the pipe 27 can be easily performed.
[0033]
【The invention's effect】
As described above, according to the system for exchanging medium of a cell culture container according to any one of claims 1 to 5, by connecting a connector to the cell culture container and operating a liquid feed pump, The flow of the culture medium is formed, and the medium can be exchanged in the cell culture container, so that the workability is excellent, and since the medium storage means and the waste medium storage means are arranged adjacent to each other, the medium The exchange does not cause a large movement of the center of gravity. In addition, a cell culture container having cells attached to the surface of a culture plate on the ground or the like by the connector is prepared, launched into space, and the medium exchange system installed in an experimental facility such as a spacecraft. , And connected to the connector, a culture test for various cells can be performed.
[0034]
According to the invention described in claim 2, a peristaltic pump having two sets of suction ports and discharge ports is used as a liquid sending pump, and the culture medium supply pipe is connected to one of the suction ports and the discharge port, Since the culture medium discharge pipe is connected to the other suction port and discharge port, the above-described flow of the culture medium in one direction can be smoothly formed. Oxygen is always supplied to the space for culturing cells by the membrane material as described above, so that additional equipment for supplying oxygen is not required, and the auxiliary equipment is simplified.
[0035]
Furthermore, according to the invention as set forth in claim 4, it is possible to simultaneously connect to the connection ends of both cell culture vessels by one connection operation by the connector, and thus exchange the medium even in a narrow test room in outer space. In doing so, the work of connecting the culture medium supply pipe and the culture medium discharge pipe to the injection hole and the discharge hole is facilitated, and according to the invention according to claim 5, even if gas is mixed into the system, In addition, the above gas can be removed by the gas separating means, and the effect of securing the stability of the system 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 system for exchanging a culture medium of FIG.
FIG. 3 is an exploded perspective view of FIG. 2;
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Main body 1a One side face 2a, 2b of main body One end 5, 6 of space part Injection / pouring hole 9 Culture plate 12 Membrane material 22 Medium storage container (medium storage means)
23 Waste medium storage container (waste medium storage means)
24, 26, 31a, 31b Connector 25 Medium supply pipe 27 Medium discharge pipe 28 Liquid supply pumps 29a, 29b Intake ports 30a, 30b Discharge ports 32 Gas separator (gas separation means)

Claims (5)

A cell culture container having a space filled with the medium, and having a medium injection hole and a medium discharge hole opened at opposing positions of the space,
Medium storage means for storing fresh medium and waste medium storage means provided adjacent to the medium storage means,
A medium discharge pipe medium supply pipe which connects the above medium housing means and said cell culture vessel and for connecting the said cell culture vessel and the waste media storage means,
Provided at the end of the medium supply pipe and the medium discharge pipe, and a connector, each of which is detachably connected to a connector provided in each of said injection hole and the Note Deana of the cell culture vessel,
A system for exchanging a culture medium for a cell culture container, comprising: 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 space of the cell culture container. . The liquid sending pump is a peristaltic pump having two sets of suction ports and discharge ports. The system for exchanging culture medium for a cell culture container according to claim 1, wherein a culture medium discharge pipe is connected. The cell culture vessel, the plate-like body the space portion medium is filled through is formed between both surfaces, the film material as an oxygen supply means for chromatic oxygen permeability covering the space portion provided And the main body is connected to the culture medium supply pipe, the injection hole for injecting the culture medium into the space, and the culture medium discharge pipe is connected 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 an injection hole is provided. The injection hole and the pouring hole of the cell culture vessel are respectively formed such that one end is opened at the opposite end of the space, and the other end is opened adjacent to the same side surface of the main body. together we are, in the connection end of the medium supply pipe and the medium discharge pipe and the connector for connecting both of the connection ends through the connector to the injection hole and the discharge hole simultaneously by a single operation The system for exchanging medium of a cell culture container according to claim 3, wherein the system is provided. The system for exchanging culture medium for a cell culture container according to any one of claims 1 to 4, wherein a gas separation means is interposed in the culture medium supply pipe.

Images