JP2009142212A - Culture solution exchanging apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culture solution exchanging apparatus that is arranged in a small-sized incubator useful in a university laboratory and exchanges culture solutions in a culture flask without being taken out from the incubator. <P>SOLUTION: The culture solution exchanging apparatus is arranged in an incubator and is used for periodically exchanging culture solutions in a culture flask in such a state that the apparatus is placed in the incubator. The apparatus includes: a tray on which a culture flask is arranged; a tray rotation mechanism for rotating the tray around a shaft vertical to the long length direction of the culture flask; a cap to be attached to the mouth of the culture flask, equipped with a discharge port for discharging the culture solution from the culture flask and an injection pipe for injecting the culture solution into the culture flask; a culture solution tank storing the culture solution; a pump for sending the culture solution from the culture solution tank through a tube to the injection pipe of the cap; a waste liquid tray for storing the culture solution discharged from the discharge port; and a control part for controlling the drives of the tray rotation mechanism and the pump. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a culture medium exchange apparatus for periodically exchanging a culture medium in a flask for cell culture.

Conventionally, cell culture of tissue pieces and the like has been performed in a culture container such as a culture flask.
General cell culture is performed by placing a culture flask in an incubator (a sterilization chamber kept in a constant environment of 37 ° C./5% CO 2 atmosphere).
In order to qualitatively provide the nutrients necessary for cell culture and to suppress the growth of germs, it is necessary to periodically exchange the culture solution in the culture flask.
Conventionally, an operator manually inserts a pipette into the flask so that the tissue pieces are not taken out together with the culture solution, but day and night, holidays, and time zones. Regardless of whether the culture medium needs to be replaced periodically, the burden on the cell culture operator is large, and in order to prevent contamination due to manual intervention during the replacement work, An exchange device is used (for example, see Non-Patent Document 1).
http://www.alliance-souyaku.co.jp/Soyaku/Jidoka/Enginering/auto-furaskobaichi.htm (Accessed on November 15, 2007)

However, in the culture flask automatic medium replacement apparatus shown in Non-Patent Document 1, the culture medium is large, and the culture liquid cannot be exchanged in the incubator. After exchanging the culture solution, the process of returning the culture flask into the incubator is performed.
During cell culture, when the cell flask is taken out from the incubator and the culture medium is exchanged in the clean bench, the culture environment (temperature, atmosphere, etc.) changes. Therefore, there is a problem as a culture medium exchange process.
Further, since the culture medium exchanger shown in Non-Patent Document 1 is large, it cannot be used in a small incubator usually used in a laboratory in a university.

  The present invention has been made in view of such circumstances, and can be placed in a small incubator used in a laboratory of a university, and the culture solution in the culture flask can be used without taking out from the incubator. It is an object of the present invention to provide a culture medium exchange device that can be exchanged.

  The culture medium exchange apparatus of the present invention is a system that is disposed in an incubator and periodically replaces the culture medium in the culture flask while being placed in the incubator, and includes a tray on which the culture flask is disposed, A tray rotating mechanism for rotating the tray with respect to an axis perpendicular to the longitudinal direction of the culture flask, a discharge hole for discharging the culture liquid from the culture flask, and an injection tube for injecting the culture liquid into the culture flask A cap to be attached to the mouth of the culture flask, a culture fluid tank in which the culture fluid is stored, a pump for feeding the culture fluid from the culture fluid tank through a tube to the injection pipe of the cap, It has a waste liquid tray for storing the culture medium discharged from the discharge hole, and a controller for controlling the drive of the tray rotating mechanism and the pump. .

  In the culture medium exchange apparatus of the present invention, the control unit drives the tray rotation mechanism at a preset time interval, and the angle at which the culture medium in the culture flask is discharged from the discharge hole to the waste liquid tray Rotate the tray to a position where the tray surface is parallel to the lower surface of the culture medium exchange device after a predetermined time has passed, and drive the pump to remove the culture tank from the culture tank. A process of injecting a culture solution into a culture flask through an injection tube is performed.

  In the culture medium exchange apparatus of the present invention, a detection unit is provided on the inner upper surface of the container of the culture medium exchange apparatus, the control unit drives a tray rotation mechanism, and the mouth of the culture flask is arranged at the lower part. End in the direction opposite to the mouth of the culture flask moves upward, the detection unit detects the end and outputs a detection signal, and the control unit outputs the detection signal according to the detection signal. The drive of the tray rotating mechanism is temporarily stopped, and the tray surface is rotated and returned to a position parallel to the lower surface of the culture medium exchange device.

  The culture medium exchange apparatus according to the present invention is arranged in the cap in a direction perpendicular to the surface of the tray, the injection tube on the upper side and the discharge hole on the lower side, and the injection tube is arranged in the culture flask. It is arrange | positioned by the length which protrudes, The said discharge hole is provided in the shape which the outer peripheral part of the mouth of the said culture flask exposes in cap vicinity vicinity.

  In the culture medium exchange apparatus of the present invention, the cap is further provided with an air hole at the top of the injection tube.

  In the culture medium exchange apparatus of the present invention, in the size of the container of the culture medium exchange apparatus, the height and depth dimensions of the container are equivalent to the diameter of a rotating circle whose diameter is the length in the longitudinal direction of the culture flask. It is characterized by its size.

  In the culture medium exchange apparatus of the present invention, the container is formed of stainless steel coated with an antibacterial coating.

As described above, according to the culture solution exchange apparatus of the present invention, the tray on which the culture flask is disposed is rotated in the direction in which the mouth of the culture flask faces downward using the tray rotation mechanism, that is, the culture flask. Because the structure that allows the culture medium to be discharged by tilting, the height and depth dimensions can be made to be the same size as the diameter of the culture flask (including the cap) and the circle around which the tray rotates. The container itself can be placed in an incubator where culture flasks of the same size as those placed on the tray are placed.
Further, according to the present invention, the height dimension of the container may be an angle at which the culture medium in the culture flask is discharged by rotating with respect to an axis perpendicular to the longitudinal direction of the culture flask. Therefore, it is possible to configure with a numerical value smaller than the diameter of the circle, for example, a dimension about the radius of the circle.

  Further, according to the present invention, since the container can be disposed in the incubator, without manually removing the culture flask from the incubator, as in the case of performing manually or in the conventional example described in Non-Patent Document 1, The culture solution in the culture flask can be exchanged, and continuous culture can be performed without changing the environment for the cells being cultured.

The present invention relates to a culture medium exchange device for automatically exchanging a culture medium when culturing adherent cells such as a tissue piece attached to the bottom of a culture flask.
Hereinafter, a culture medium exchange apparatus according to an embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a conceptual diagram showing a structure of a culture medium exchange device as one configuration example of the embodiment as viewed from the side. FIG. 2 is a conceptual diagram showing the structure of the culture medium exchange apparatus shown in FIG. 1 as viewed from the front. Here, the container surface parallel to the surface of the circle formed by rotation of the culture flask is taken as the container side surface shown in FIG. 2, and the container surface facing the cap of the culture flask in FIG. 2 (right surface in FIG. 2) 1 is the front of the container shown in FIG. 1, the surface of the container facing in parallel with the front of the container is the back of the container, the surface in contact with the ground is the bottom of the container, and the surface of the container facing in parallel with the bottom of the container The upper surface is the container upper surface.

The container of the apparatus is opposed to the container side plate 2 provided perpendicularly to the container bottom plate 1 and in parallel with each other at a distance D1 (height described later) in parallel to the container bottom plate 1. The container upper plate 3 is provided, the container front plate 4 provided perpendicular to the container lower plate 1, and the container back plate 5 provided parallel to the container front plate 4. .
The container front plate 4 and the container back plate 5 are fixed to the container top plate 3 through hinges T so as to be freely opened and closed. The container lower surface plate 1, the container side surface plate 2, the container upper surface plate 3, the container front surface plate 4, and the container rear surface plate 5 described above are formed of stainless steel coated with an antibacterial material in the present embodiment. However, the container bottom plate 1, the container side plate 2, the container top plate 3, the container front plate 4, and the container back plate 5 may be antibacterial materials such as materials that can be sterilized with alcohol such as polystyrene, polypropylene, and polycarbonate. Can be used.

  Inside the container are a tray 11 on which the culture flask B is mounted, a tray rotating mechanism section 12 that rotates the tray 11, a culture medium tank 13 that stores the culture medium to be injected into the culture medium flask B, and an inside of the culture flask B. The culture medium is stored in the culture flask B through the waste liquid tray 14 for storing the culture liquid as the waste liquid discarded from the culture medium and the culture liquid tank 13 through the tubes 21 and connectors 50 (connectors for distributing the tubes 21 to the culture flasks B). A pump 15 and the like for injecting the liquid are provided.

The tray 11 is fixed to a shaft 16 that is rotated by the tray rotating mechanism 12 by a fixing member 17 and is rotatably supported by a fixing member 18 provided on the container side plate 2. As a result, the tray 11 rotates with respect to the shaft 16 at an angle at which the tray rotation mechanism unit 12 rotates the shaft 16. The surface of the circle C1 formed by this rotation is parallel to the container side plate 2.
The culture flask B is mounted on the tray 11 by being fixed by a fixture 19 so that the longitudinal axis is parallel to the surface of the circle C1 and the bottom is parallel to the surface of the tray 11. The Therefore, a circle C2 formed by the culture flask B rotating together with the tray 11 is also parallel to the container side plate 2 like the circle C1.

In the present embodiment, the rotation direction H of the tray 11 is the rotation direction of the clock hand because the cap 20 attached to the mouth of the culture flask B is located on the right side in FIG.
However, if the cap 20 attached to the mouth of the culture flask B moves downward from a position where the surface of the tray 11 is parallel to the container lower surface plate 1, the culture medium in the culture flask B is discharged. For this reason, the rotation direction H is set in the direction in which the cap 20 moves downward.
As shown in FIG. 2, the size of the quadrangular surface (rectangle formed by the height D1 and the depth D2) of the container side plate 2 of the container is set to a size in which the circle C2 is inscribed, The diameter of the circle C2 is set to be the length of the side of the square.
Therefore, the culture medium exchange apparatus of the present embodiment is usually used because the size of the container corresponds to the length in the lengthwise direction of the culture flask B and the height and depth are substantially the same. If it corresponds to a culture flask, it can be inserted into an incubator that is usually used. The lateral width is the lateral width × the number of culture flasks B arranged in the culture medium exchange device, and needs to correspond to the lateral width of the incubator internal space.

The rising position detector 25 detects the rotation of the tray 11 to the angle (preset) at which the culture medium inside is completely discharged from the culture flask B. Is provided at a position where the end of the culture flask facing the cap 20 of the culture flask B on the long axis is detected.
The descending position detector 26 is provided at a position on the circle C <b> 2 on the container back plate 5 in order to detect a position where the tray 11 is parallel to the container bottom plate 1.
As the raised position detector 25 and the lowered position detector 26, proximity switches, optical sensors, toggle switches, and the like can be used.
Since the tray 11 rotates and the culture solution is discharged from the culture flask B, the waste solution tray 14 is disposed at a position on the circle C1 and the circle C2 on the container lower surface plate 1, and previously contains cresol or the like. Inject the fungicide.
The culture medium tank 13 and the tank 15 are disposed so as to overlap the lower part of the tray 11, the tray 11 rotates in the rotation direction RH opposite to the rotation direction H, and the surface of the tray 11 is parallel to the container lower surface plate 1. It is arranged to be adjusted to the height as a stopper that stops at a certain position.

The cap 20 is a part of the culture medium exchange apparatus according to the present embodiment, and is attached to the mouth of the culture flask B. As shown in FIG. 3, the air hole 201, the injection hole 202, and the discharge hole are formed in the top surface 20a. Three holes 203 are opened in the order of an air hole 201, an injection hole 202, and a discharge hole 203 from the top along an axis perpendicular to the container lower surface plate 1. 3A is a front view of the culture flask B equipped with the cap 20, and FIG. 3B is a side view.
The injection hole 202 is provided with the tube 21 and the inner tube 22. The tube 21 is connected to the pump 15. The pump 15 injects the culture solution sucked from the culture solution tank 13 into the culture flask B through the tube 21.

The discharge hole 203 is provided in the vicinity of the outer peripheral portion of the top surface 20a so that the outer peripheral portion of the mouth of the culture flask B is exposed, that is, provided at a position where the waste liquid is easily discharged when tilted together with the tray 11. ing.
The air hole 201 is provided to allow air to flow into the culture flask B from the outside so that the internal pressure becomes low when the culture liquid is discharged from the discharge hole 203 and the discharge of the waste liquid is not delayed. The air hole 201 is provided to discharge the air in the culture flask B to the outside so that the internal pressure becomes high when the culture liquid is injected from the injection hole 202 and the injection of the culture liquid is not delayed. ing. The air hole 201 is also opened so that, for example, a 5% CO ₂ atmosphere (atmosphere) as an environment in the incubator is supplied into the culture flask B.

The inner tube 22 has a length of about ½ of the length of the culture flask B in the longitudinal direction, and has an angle θ with respect to the bottom of the culture flask B in the culture flask B and has a cap 20. Is attached.
As a result, the injected culture solution flows into the entire container from the vicinity of the center of the culture flask B. By providing the angle θ described above, the position at which the culture solution is injected into the bottom of the culture flask B becomes closer, so that the tissue piece attached to the bottom of the culture flask B is peeled off by the pressure at which the culture solution is injected. Can be suppressed.

Here, the angle θ and the length of the inner tube 22 are set so that the culture solution extraction port 22a of the inner tube 22 is located at a distance d from the bottom of the culture flask B. This distance d is a distance at which the liquid level P of the culture solution does not contact the extraction port 22a when the tray 11 rotates.
The tube 21 once exits the container from the injection hole 202 of each cap 20 through the opening 23 provided in the container upper surface plate 3, and the container side plate 2 in the vicinity where the culture medium tank 13 is disposed. The inside of the container enters through the opening 24 provided in the, and is connected to the pump 15.

Next, the operation of the culture medium exchange apparatus shown in FIGS. 1 and 2 will be described. Here, a control unit (not shown) is disposed inside or outside the container, and this control unit controls the driving and stopping of the tray rotation mechanism unit 12 and the pump 15.
First, the culture medium exchange apparatus of this embodiment is sterilized with 70% alcohol or the like, and after injecting the culture liquid into the culture flask B, the cap 20 is attached, the tube 21 is attached to the injection hole 202, and then the culture is performed. Set the liquid exchange cycle (exchange time), for example, exchange time data for exchanging the culture solution every 6 hours is set in the control unit, and the culture solution exchange device is placed in the incubator. Initiate cell culture.

A. The control unit outputs a rotation drive signal to the tray rotation mechanism unit 12 when the count time data of the internal timer matches the exchange time data.
The tray rotation mechanism unit 12 starts to rotate in the rotation direction H when the rotation drive signal is input.
As a result, the tray 11 rotates, and the culture flask B becomes inclined as the angle formed between the axis perpendicular to the container lower surface plate 1 and the axis perpendicular to the surface of the tray 11 becomes perpendicular. The culture solution in B is discharged as waste liquid from the discharge hole 203 to the waste tray 14.

B. When the rising position detector 25 detects the end of the culture flask B that moves up and approaches the container upper surface plate 3, it outputs an upper position detection signal to the control unit.
When the upper position detection signal is input, the control unit outputs a rotation stop signal to the tray rotation mechanism unit 12.
At this time, the angle formed between the axis perpendicular to the container lower surface plate 1 and the axis perpendicular to the surface of the tray 11 is almost a right angle, and the culture solution in the culture flask B is completely discharged to the waste tray 14.
As a result, the tray rotation mechanism unit 12 stops for a certain period of time in a state where the angle between the axis perpendicular to the container lower surface plate 1 and the axis perpendicular to the surface of the tray 11 is perpendicular.

C. The control unit outputs a reverse rotation drive signal to the tray rotating machine rear part 12 after a predetermined time has elapsed after outputting the rotation stop signal.
When the tray rotation mechanism 12 receives the reverse rotation drive signal, the tray rotation mechanism 12 starts to rotate in the rotation direction RH in which the surface of the tray 14 is parallel to the container lower surface plate 1.

D. When the descending position detector 26 detects the end of the culture flask B as the tray 14 descends and approaches the upper portion (stopper) of the pump 15, it outputs a lower position detection signal to the control unit.
When the lower position detection signal is input, the control unit outputs a reverse rotation stop signal to the tray rotation mechanism unit 12.
At this time, the angle formed between the axis perpendicular to the container lower surface plate 1 and the axis perpendicular to the surface of the tray 11 is substantially 0, the surface of the tray 11 and the container lower surface plate 1 are in a parallel position, and the tray 14 is pumped. 15 is brought into close contact with the upper portion.

E. The control unit outputs the reverse rotation signal and outputs a pump driving signal for driving the pump 15 to the pump 15.
When the pump drive signal is input, the pump 15 sucks out the culture solution from the culture solution tank 13, sends it to the tube 21, and injects the culture solution into the culture flask B.
The control unit counts the time after the pump drive signal is output, and outputs a pump drive stop signal to the pump 15 when detecting that a preset injection time has elapsed. The injection time is a time actually measured in advance, and is set as a time during which a necessary culture solution is injected into the culture flask B.

F. When outputting the pump drive stop signal, the control unit resets the count time data of the internal timer and starts counting a new count time.
By repeating the treatments A to F described above, the culture medium can be exchanged at a necessary cycle without taking out the culture medium exchange device from the incubator, that is, without changing the culture environment of the culture flask B, Since no human intervention is required, cells of tissue pieces can be easily cultured in the incubator.
In addition, since the culture medium exchange apparatus of the present embodiment has a height x depth that is close to the length of the culture flask in the longitudinal direction, if it is an incubator using a culture flask mounted on the apparatus, it can be used without modification. It can be arranged, and can be used not in a large incubator used by a pharmaceutical company but in a commercially available small incubator used in a university laboratory, and cell culture can be easily automated.

For example, the culture flask B shown as an example in the present embodiment is a medium size (25 cm ² ), has a height including the cap 20 of 28.8 mm, a length in the longitudinal direction of 109 mm, and a width of 40 mm. For this reason, as for the size of the container of the culture medium exchange device, height (D1) × depth (D2) × width (D3) is 140 mm × 154 mm × 306 mm.
In this embodiment, since six culture flasks are mounted on the tray 11 in parallel, the horizontal width is 306 mm, but the horizontal width varies depending on the number of culture flasks mounted.
The size of the internal space of an incubator for culturing a culture flask of this size is a commercially available one that is almost 200 mm high, 230 mm deep and 310 mm wide (Shibata Kagaku Co., Ltd., Compact Incubator, SCI-13). Therefore, the culture medium exchange device of the present embodiment can be arranged without any problem.

When a large-sized (225 cm ² ) culture flask is used, the length in the longitudinal direction including the cap 20 is 204 mm, the height is 49 mm, and the width is 125 mm. For this reason, as for the size of the container of the culture medium exchange device, for example, height (D1) × depth (D2) × width (D3) is 245 mm × 255 mm × 250 mm.
In this case, two culture flasks are mounted on the tray 11 in parallel, and the horizontal width is 250 mm, but the horizontal width varies depending on the number of culture flasks mounted.
The size of the internal space of the incubator for culturing culture flasks of this size is about 350 mm in height, 350 mm in depth, 350 mm in width (Daiwa Kagaku Co., Ltd., IJ300W). The culture medium exchange device of the embodiment can be arranged.

It is the conceptual diagram seen from the front of the apparatus explaining the structural example of the culture solution exchange apparatus by one Embodiment of this invention. It is the conceptual diagram seen from the side of the apparatus explaining the structural example of the culture solution exchange apparatus by one Embodiment of this invention. It is a conceptual diagram which shows the structural example of the cap 20 with which the culture flask of the culture solution exchange apparatus by one Embodiment of this invention is mounted | worn.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Container lower surface plate 2 ... Container side surface plate 3 ... Container upper surface plate 4 ... Container front surface plate 5 ... Container rear surface plate 11 ... Tray 12 ... Tray rotation mechanism part 13 ... Culture liquid tank 14 ... Waste liquid tray 15 ... Pump 16 ... Shaft 17 , 18 ... Fixing member 19 ... Fixing tool 20 ... Cap 21 ... Tube 22 ... Internal pipe 23, 24 ... Opening 25 ... Ascending position detector 26 ... Ascending position detector 50 ... Connector 201 ... Air hole 202 ... Injection hole 203 ... Discharge hole

Claims (7)

It is a culture medium exchange device that is arranged in an incubator and periodically replaces the culture medium in the culture flask while being placed in the incubator.
A tray on which the culture flask is placed;
A tray rotating mechanism for rotating the tray with respect to an axis perpendicular to the longitudinal direction of the culture flask;
A cap attached to the mouth of the culture flask provided with a discharge hole for discharging the culture liquid from the culture flask and an injection tube for injecting the culture liquid into the culture flask;
A culture medium tank in which the culture medium is stored;
A pump for feeding the culture solution from the culture solution tank through a tube to the injection tube of the cap;
A waste liquid tray for storing the culture medium discharged from the discharge hole;
A culture medium exchange device comprising: a tray rotation mechanism and a control unit that controls driving of the pump.   The control unit drives the tray rotation mechanism at a preset time interval, rotates the tray to an angle at which the culture medium in the culture flask is discharged from the discharge hole to the waste liquid tray, and is set in advance. After the elapsed time, the tray surface is rotated back to a position parallel to the lower surface of the culture medium exchange device, and the pump is driven to enter the culture flask from the culture tank through the injection tube. 2. The culture medium exchange apparatus according to claim 1, wherein a process for injecting the culture medium is performed. A detection unit is provided on the inner upper surface of the container of the culture medium exchange device,
When the control unit drives a tray rotation mechanism and rotates the tray in a direction in which the mouth of the culture flask is at the bottom, the end opposite to the mouth of the culture flask moves upward, and the detection unit Detects the end and outputs a detection signal, the control unit temporarily stops driving the tray rotating mechanism by the detection signal, and the tray surface is parallel to the lower surface of the culture medium exchange device The culture medium exchange apparatus according to claim 2, wherein the culture medium exchange apparatus is rotated back to a position.   In the cap, in a direction perpendicular to the surface of the tray, the upper side is arranged as the injection tube on the upper side and the lower side as the discharge hole, and the injection tube is arranged with a length protruding into the culture flask, The culture medium exchange apparatus according to any one of claims 1 to 3, wherein the discharge hole is provided in a shape in which an outer peripheral portion of the mouth of the culture flask is exposed in the vicinity of the outer peripheral portion of the cap. .   5. The culture medium exchange apparatus according to claim 4, wherein an air hole is further opened in the cap above the injection tube.   The size of the container of the culture medium exchange device is characterized in that the height and depth of the container are equivalent to the diameter of a rotating circle whose diameter is the length in the longitudinal direction of the culture flask. The culture medium exchange apparatus in any one of Claims 1-5.   7. The culture medium exchange apparatus according to claim 6, wherein the container is made of antibacterial coated stainless steel.

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