JP2009100652A - Fluid feeding device and cell culture apparatus using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluid feeding device having a simple constitution, and to provide a cell culture apparatus that uses the device. <P>SOLUTION: A rotor 1 of a stirrer is rotatably housed in a housing chamber 2, and a suction port 3 and a discharge port 4 for making a fluid go in and out are provided in the housing chamber 2. The discharge port is provided at a position where the fluid can be made to discharge from the discharge port to the outside of the housing chamber by the inertial force produced in the fluid in the housing chamber in the inner sidewall of the housing chamber. The suction port is provided at (a) a position where only the force that causes inflow as a result of the discharge from the discharge port would operate, or at (b) a position where a larger force that causes inflow as a result of discharge from the discharge port operates, even though the force operates so as to discharge the fluid by the inertial force produced in the fluid in the housing chamber, when the rotor is rotated. Furthermore, the compact cell culture apparatus is obtained, by connecting a culture flow channel to the suction port and the discharge port of the fluid feeding device. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a fluid feeder that generates a fluid flow in a certain direction, a cell culture device using the fluid feeder, a cell culture method using the cell culture device, and a drug metabolism simulation method.

  Ordinary animal cells have wall-adhesive properties. If they do not attach to anything, they cannot survive as well as proliferate and function. For this reason, a single-layer culture (two-dimensional cell culture) method in which cells are attached to a culture substrate such as polystyrene subjected to a cell adhesion treatment is usually used. On the other hand, in recent years, three-dimensional cell culture technology has attracted attention. Three-dimensional cell culture is a cell culture performed in a three-dimensional environment, which improves the functions of the cells themselves and enables the culture of tissues and cells with functions found in organs and organs. (For example, non-patent documents 1 to 4). However, it is also possible to perform culture that is the same as three-dimensional cell culture even in two-dimensional cell culture by using a functional culture substrate or by optimal design of the culture medium (for example, Patent Documents 1 and 2).

In order to perform cell culture, large-scale culture plants have been developed by pharmaceutical companies and the like. On the other hand, a relatively small-scale cell culture system is important at the level of research and development. Examples of such a relatively small system include the following.
Hollow fiber bioreactor: A product manufactured by SciMedia is known, which supplies nutrients using a hollow fiber membrane and is composed of an incubator, a culture instrument, electrodes, various pumps, and the like.
Cell Cube System for Mass Culture: A product made by Corning is known, and is an apparatus constituted by a system controller, a reflux pump, a medium pump, an oxygen generator, and the like.
Membrane bioreactor: A product made by Sartorius is known, which enables mass culture by rotating an incubator.
RCCS (Rotary Cell Culture System): One of the three-dimensional culture systems known from Synthecon, which rotates the culture vessel itself using a rotation control device in a normal incubator. , PH and oxygen electrodes are not required.
In addition, there is a culture plate product that forms a cell mass (cells are three-dimensionally cultured in the cell mass) by special treatment, but it does not flow the culture solution, so it can maintain long-term culture and cell function. It is difficult. In addition, 3D culture tissue construction plate products using extracellular matrix, CELLine flask (BD) capable of producing antibodies and recombinant proteins, etc. The product is being developed.

  Culture technology for various cell types can dramatically improve the ability of cells themselves to form and repair tissues, or the ability to produce substances such as medical proteins, so artificial organs and stem cell treatment in the field of regenerative medicine Furthermore, it has attracted much attention as a biotool for producing protein for medical use and developing biodevices (medical instruments incorporating cells).

However, conventional culture devices have been developed for specific cell types such as cells producing protein for medical use, and such dedicated devices are difficult to observe due to anchorage and cell observation of various cells. For some reasons, stress on cells due to a high flow rate, and the use of a large amount of expensive culture solution, it cannot be applied to various cell types such as embryonic stem cells, neural stem cells, and primary cultured cells.
In order to process a large number of tests at once, a large number of devices must be arranged in parallel. However, this is not easily realized from the viewpoint of the occupied area of the device and the cost.

Therefore, if a culture device that can be miniaturized with a simple configuration and a culture device that is inexpensive can be developed, various cell types can be cultured in a three-dimensional environment, and many test processes can be performed in parallel. And will be able to contribute greatly to the development of culture technology.
In this regard, the present inventors have examined a conventional culture device, and the conventional device mainly has a combination of a pump device for feeding a culture solution and a tube. It was found that there was a problem.
(A) In the structure which combined the pump apparatus and the tube, the whole apparatus must be complicated and large, and the price of the apparatus itself is also expensive.
(B) For multi-sample processing, many pumps and tubes must be arranged in parallel, which further increases the occupation area and cost of the apparatus.
(C) A large amount of culture solution is required to fill the inside of the pump and tube.
(D) Bubbles are easily generated by the pump pressure, and the culture volume is reduced by the bubbles or the pump does not operate normally. To solve this, the flow rate of the culture solution is kept high, and the culture layer or Air must be evacuated from the tube. However, in normal cells, stem cell cultures, and tissue cultures, cells are damaged when the flow rate is increased.

  The above problems are not only in cell culture, but also in fluid chemical tests using microreactors, interaction tests between biomolecules such as nucleic acids, proteins, sugars, and lipids, enzyme reaction tests, chemical synthesis, binding reactions The same problem exists in tests, drug metabolism tests, and the like.

Simulating in vivo drug metabolism is extremely important in evaluating drug efficacy and side effects at the drug development stage, but it is useful in vitro to reproduce drug metabolism in vivo. No evaluation system has yet been obtained. As a small-scale evaluation system capable of achieving high throughput, stationary culture using a multiwell plate is the best, but it is far from predicting drug metabolism behavior in vivo. Therefore, an in vitro cell function evaluation system capable of high throughput that is sufficient for simulating drug metabolism behavior in vivo is eagerly desired.
JP 2004-166717 A JP 2004-267207 A Bell E, IvarssonB, Merrill C (1979) Proc Natl AcadSciUSA. Mar; 76 (3): 1274-8. Chen SS, RevoltellaRP, Papini S, Michelini M, Fitzgerald W, Zimmerberg J, MargolisL (2003) Stem Cells. 21 (3): 281-95. Saito N, Okada T, Horiuchi H, Murakami N, Takahashi J, NawataM, Ota H, Nozaki K, Takaoka K. (2001) Nat Biotechnol. Apr; 19 (4): 332-5. Evans GR, Brandt K, Widmer MS, Lu L, MeszlenyiRK, Gupta PK, Mikos AG, Hodges J, Williams J, GurlekA, Nabawi A, Lohman R, Patrick CW Jr. (1999) Biomaterials. Jun; 20 (12): 1109-15.

  An object of the present invention is to solve the above-described problems, provide a fluid feeder having a simple configuration, and provide a cell culture device using the fluid feeder. A further object of the present invention is to provide a cell drug metabolism simulation system that is superior to the conventional one using the cell culture apparatus.

  As a result of intensive studies to achieve the above-mentioned object, the present inventors have created a fluid flow in a certain direction by utilizing the inertial force of the fluid around the rotation drive unit generated by the rotation of the stirrer. It succeeded in obtaining cell culture results superior to static culture in a simple and space-saving manner. Furthermore, when cell culture using the fluid feeder is applied to a drug metabolism simulation, it has been found that a marked drug clearance can be obtained as compared with the prior art, and the present invention has been completed.

That is, the present invention
[1] The stirrer rotor has a storage chamber in which the rotor is rotatably stored, and the storage chamber is provided with a suction port and a discharge port for allowing fluid to enter and exit,
The discharge port is provided on the inner side wall of the storage chamber at a position where the fluid can flow out of the storage chamber from the discharge port due to inertial force generated in the fluid in the storage chamber when the rotor is rotated.
When the rotor is rotated, the suction port
(B) A position where only the force to flow in due to outflow from the discharge port acts, or
(B) Although the force of the fluid trying to flow out is exerted by the inertial force generated in the fluid in the storage chamber, the force of flowing in due to the outflow from the discharge port acts more than that force. It is provided in such a position,
Thereby, when the rotor is rotated, a fluid feeding action in which the fluid outside the storage chamber flows into the storage chamber from the suction port and flows out of the storage chamber from the discharge port,
Fluid feeder,
[2] The fluid feeding device according to [1], wherein the discharge port is provided at a position including at least a portion surrounding a rotation circle drawn by rotation of the rotor, on the inner side wall of the storage chamber.
[3] The fluid feeding device according to the above [1], wherein the discharge port is provided at a position not including a portion surrounding a rotation circle drawn by rotation of the rotor on the inner side wall of the storage chamber.
[4] The fluid feeding device according to the above [1], wherein the suction port is provided on the inner side wall of the storage chamber;
[5] When the suction port is assumed to be a straight or arc-shaped flow path connecting the suction port and the discharge port, the flow path is positioned so as to be parallel to the wall in the rotation axis direction of the rotor. The fluid feeding device according to [4], wherein the fluid feeding device is provided and the center axis of the flow path does not pass through the rotation center of the rotor;
[6] The above-mentioned [5], wherein the rotation circle drawn by the rotation circle of the rotor circumscribes the side surface of the flow path connecting the suction port and the discharge port, or crosses one side surface but does not cross the other side surface. Fluid feeding device;
[7] The above [6], wherein the internal side wall is inscribed in the side surface of the flow path when the internal side wall of the storage chamber is assumed to extend into the flow path along a rotation circle drawn by rotation of the rotor. A fluid feeder as described;
[8] The above [2], wherein the suction port is provided at a position different from the discharge port and not including a portion surrounding the rotation circle drawn by rotation of the rotor on the inner side wall. A fluid feeder as described;
[9] The fluid feeding device according to any one of [1] to [8], wherein the stirrer rotor is housed in the housing chamber so as to be horizontally rotatable.
[10] Any of the above [1] to [9], wherein the stirrer is a magnetic stirrer, and the rotor is rotated by the action of magnetism emitted from a stirrer main body driving device arranged outside the storage chamber. A fluid feeder according to claim 1;
[11] A cell culture apparatus comprising the fluid feeder according to any one of [1] to [10] above and a culture channel provided outside a storage chamber of the fluid feeder,
One end of the culture channel is connected to the inlet of the fluid feeder, and the other end is connected to the outlet of the fluid feeder,
A cell culture device, wherein the culture solution is configured to pass through a culture flow path by a fluid feed action of the fluid feed device;
[12] The stirrer of the fluid feeding device is a magnetic stirrer, and the rotor rotates horizontally by the action of magnetism emitted from the stirrer body driving device disposed outside the storage chamber,
The stirrer main body driving device is configured to be capable of rotating a plurality of rotors by one unit.
A plurality of units including a configuration in which the fluid feeding device and the culture channel are connected are arranged on the stirrer body driving device, and the plurality of units are operated by one stirrer body driving device. The cell culture device of [11] above;
[13] The cell culture device according to [11] or [12] above, which is a drug metabolism simulator for cells;
[14]
A culture substrate is disposed in the culture channel of the cell culture device described in [11] or [12] above, a medium is added to the culture channel and the chamber of the fluid feeder, the cells are seeded, and the cells are seeded. A cell characterized in that, after being held in or on the surface of the culture substrate, the medium is circulated in the culture channel in a certain direction by rotating the stirrer rotor of the fluid feeder. A culture method; and [15] a culture substrate is disposed in the culture flow path of the cell culture device according to [11] or [12] above, a medium is added to the culture flow path and the accommodation chamber of the fluid feeder, and the cell The medium containing the drug is retained in or on the surface of the culture substrate, and then the drug-containing medium is circulated by rotating the stirrer rotor of the fluid feeder to change the drug concentration. Measuring cells, including measuring Simulation method of things metabolism;
I will provide a.

  Originally, the Stirrer rotor is a member for stirring the liquid, so that if the rotor is simply rotated as it is in the liquid, only a spiral flow is generated in the liquid tank. On the other hand, in the fluid feeding device of the present invention, as shown in FIG. 1, a stirrer rotor 1 and a storage chamber 2 are combined, and specific openings 3 and 4 are added thereto, thereby providing a constant like a pump. It causes fluid feeding action (intake and discharge) in the direction. This fluid feeding action in a certain direction is fundamentally different from the omnidirectional stirring action by the stirrer rotor.

The generation principle of the fluid feeding action by the fluid feeding device is as follows.
That is, as shown in FIG. 1, a stirrer rotor 1 is rotatably accommodated in an accommodation chamber 2, and a suction port 3 and a discharge port 4 are provided in the accommodation chamber 2. Here, when the rotor is rotated, the fluid receives a force in the radial direction and the circumferential direction of the rotation circle, but the flow from the discharge port 4 to the outside of the storage chamber becomes dominant due to the inertial force, and as a result, the fluid flows into the suction port 3. Thus, a fluid feeding action of flowing into the storage chamber and flowing out of the storage chamber from the discharge port 4 occurs, and a flow in a certain direction is obtained.

The fluid feeding device according to the present invention is a device that creates a fluid flow (fluid feeding action) in a certain direction by using the inertia force of the fluid generated by the rotation of the stirrer rotor. By using magnetic force etc., this device can flow the culture medium in a non-contact manner with the drive device main body, and can be easily distributed and cultured, and the risk of contamination is greatly reduced. There is also an effect.
Furthermore, flow culture on the same scale as stationary culture using a multi-well plate conventionally used for multi-sample evaluation is possible, and cell function evaluation and the like can be performed with high throughput.

Below, the structure of the fluid feeder of this invention is demonstrated.
FIG. 1 is a schematic view showing an example of the fluid feeding device, and schematically shows the internal structure and operating principle of the device. FIG. 1 is a top view of the apparatus. There may be a top plate, but it is not essential and is not drawn here to show the interior of the containment room. The rotation center 1a of the rotor is added for easy understanding of the drawing, and does not need to be an actual rotation shaft. FIG. 2 shows a cross-sectional view of the device when cut in the XY direction of FIG.
As shown in FIGS. 1 and 2, the fluid feeder includes a storage chamber 2 in which a stirrer rotor 1 is rotatably stored. The storage chamber 2 is provided with a suction port 3 and a discharge port 4 for allowing fluid to enter and exit. In the example of the figure, both the suction port 3 and the discharge port 4 are the inner side walls of the storage chamber 2 (that is, the rotation circle drawn by the rotation of the rotor 1 or the rotation circle and its extension in the direction of the rotation axis). Wall 1b) 2a.
The reason why the discharge port acts as an opening for discharging fluid is the inertial force of the fluid generated by the rotation of the rotor, as described above. That is, when the rotor 1 is rotated, the fluid flowing from the discharge port 4 to the outside of the storage chamber becomes dominant due to the inertial force of the fluid in the storage chamber. As a result, the fluid flows into the suction port as indicated by the arrow L1. 3 flows into the storage chamber and flows out of the storage chamber from the discharge port 4 as indicated by an arrow L2.
The direction of the flow path connected to the discharge port (that is, the flow path from the discharge port to the outside of the storage chamber) is not particularly limited. However, as shown in FIG. It is possible to efficiently use the tangential inertia force of the fluid that rotates and efficiently as the discharge force.

The stirrer used in the apparatus may be a shaft drive type stirrer in which a driving source such as a motor and a rotor are directly connected by a rotating shaft. However, if a magnetic stirrer is used, the rotating shaft and the shaft are allowed to pass therethrough. A through-hole or a bearing structure can be omitted, and the configuration of the apparatus becomes simpler. Moreover, since there is no contact with outside air, the risk of internal contamination by various bacteria is extremely low.
In the following description, the present invention will be described using an embodiment in which a magnetic stirrer is used as a stirrer. However, the present invention may be appropriately replaced with another driving type such as a shaft drive type stirrer.

As shown in FIG. 11A, the magnetic stirrer itself is a known stirrer configured to rotate a rotor (also referred to as a stirrer) 1 by the action of a magnetic field generated from a stirrer main body driving device S. is there.
On the upper surface of the stirrer body driving device S, a stage S1 for placing a tank to be stirred is provided, and a magnetic field generator is accommodated below the stage S1. Usually, the rotation plane of the rotor is a plane parallel to the surface of the stage S1.
In which direction the surface of the stage S1 of the stirrer body drive device S is directed (upward on the horizontal surface, downward on the horizontal surface, vertical surface, upward or downward with an arbitrary inclination, etc.) The direction in which the rotation center axis of the child is directed is free. In the following description, unless otherwise specified, the present invention will be described with an aspect in which the surface of the stage S1 is upward or downward on a horizontal plane and the rotor is rotated horizontally. The aspect in which the rotor is rotated horizontally simplifies the apparatus configuration, and the features of the present invention are most remarkable. You may change suitably the inclination of the surface of stage S1, and the inclination of the rotation center axis | shaft of a rotor.
The rotor is usually stable to the fluid to be stirred and has an outer layer made of a material with excellent wear resistance (for example, Teflon (registered trademark), polystyrene, glass, polypropylene, polyethylene, etc.), The interior has a configuration in which a permanent magnet or a magnetic material is embedded so that it can be rotated by the action of a magnetic field generated from the stirrer main body driving device S.
The magnetic stirrer of the figure is a single type in which one rotor 1 is rotated by one stirrer body driving device S, but a plurality of rotors can be simultaneously rotated on the stage of one stirrer body driving device. Multi-types may be configured and used.

The shape of the rotor is not particularly limited, and a conventionally known magnetic stirrer rotor may be used, but a shape that generates a flow around the outer circumference of a circle drawn by rotation is preferable. Examples of such a shape include a rod shape, a cross shape, and a radial shape. Moreover, the center part of the trunk | drum may swell so that rotation may become smooth.
When the rotor has a rod shape, the cross-sectional shape perpendicular to the longitudinal direction may be a circle, a triangle, a rectangle, a polygon, or the like. The same applies when the rotor has a cross shape, a radial shape, or the like.
The diameter of the rotation circle drawn by the rotation of the rotor (the circle of the outline of the circular area occupied by the rotor when the rotation of the rotor is viewed from above) is not particularly limited, but is 0.1 mm. If it is -200 mm, especially 1 mm-10 mm, the said fluid feeder itself and by extension the cell culture apparatus using it will become compact, and it will become possible to arrange many said culture apparatuses in parallel simultaneously.

The number of rotations of the rotor is the diameter of the rotation circle, the shape of the rotor, the arrangement of the inlet and outlet (ie, where in the culture channel the storage chamber is located), the type of cells to be cultured, etc. However, it is preferably about 1 to 1000 revolutions per minute, particularly about 10 to 100 revolutions per minute.
Further, the rotation direction of the rotor may be clockwise or counterclockwise.

The size of the storage chamber of the fluid feeder is not limited as long as it can rotatably store the stirrer rotor. However, if the size of the storage chamber is excessively large with respect to the rotation circle, the suction port and the discharge port may be used. Since the outlet is far away from the rotor, it is difficult to obtain a fluid feeding action by inertial force.
From such a point, as shown in FIG. 3, the gaps g1, g2, and g3 between the rotating circle 1b and the inner side wall surface are about 0.1 mm to 10 mm, and particularly preferably 0.2 mm to 5 mm.

  The basic shape when the storage chamber is viewed from the direction of the rotation axis of the rotor is a shape that can fit the rotation circle appropriately, such as a circle as shown in FIG. 3A or a rectangle as shown in FIG. preferable. In particular, the circular shape is a preferable shape because the gap between the rotating circle and the inner side wall surface is uniform over the entire circumference, and the rotor can be held on the outer circumference without causing unintended turbulence.

As shown in FIG. 3A and the like, by making the chamber shape when the storage chamber is viewed from the direction of the rotation axis into a circular shape adapted to the rotation circle, the rotor does not have a rotation shaft at the center. It is held on the wall surface and can be rotated stably. Such an aspect is preferable from the viewpoint of a simpler configuration. Although it depends on the diameter of the rotating circle, it is preferable that the difference between the diameter of the storage chamber and the diameter of the rotating circle is about 0.5 mm to 2 mm without backlash.
Further, as shown in FIG. 3B, when the interior shape of the storage chamber is a square when viewed from the direction of the rotation axis, for example, if the internal rotor freely moves at the time of stoppage, It may be difficult to start smoothly. In such a case, a rotation shaft may be provided as appropriate along the rotation center axis of the rotor.

The discharge port 4 of the fluid device is provided in the inner side wall 2a at a position where the fluid can flow out of the storage chamber from the discharge port 4 due to inertial force generated in the fluid in the storage chamber when the rotor is rotated. It only has to be. In a preferred embodiment, as shown in FIG. 4A, the discharge port 4 includes at least a part 2a3 of the part 2a1 surrounding the rotation circle drawn by the rotation of the rotor 1 of the inner side wall 2a. Provided. In Fig.4 (a), the discharge outlet 4 is extended to the part 2a2 surrounding the extension of the rotating circle direction of a rotation circle of an internal side wall. By providing the discharge port at such a position, when the rotor is rotated, the fluid can flow out of the storage chamber from the discharge port due to inertial force generated in the fluid in the storage chamber. In FIG. 4A, only a part 2a3 of the inner wall 2a surrounding a rotation circle drawn by the rotation of the rotor 1 is opened as a discharge port. The entire shape may be open, that is, the bottom of the storage chamber 2 and the bottom of the discharge port 4 may be flat. Although the fluid level of the fluid is not shown in FIG. 4 (a), the height of the fluid level may be such that the rotor may be completely hidden or a part of the rotor may protrude from the fluid level. Good.
In another embodiment, as shown in FIG. 6 (d), the discharge port 4 is provided at a position on the inner side wall 2 a that does not include a portion surrounding the rotation circle drawn by the rotation of the rotor 1. Even in such a case, while the rotor rotates clockwise, the fluid flows out of the storage chamber from the discharge port due to the inertial force generated in the fluid in the storage chamber, while the fluid is generated by the inertial force generated in the fluid in the storage chamber. Because the force that the fluid tries to flow into the suction port from outside the storage chamber due to the outflow from the discharge port is larger than the force that tries to flow out from the suction port, as a result, when the rotor is rotated Thus, a fluid feeding action occurs in which the fluid outside the storage chamber flows into the storage chamber from the suction port and flows out of the storage chamber through the discharge port.

The suction port of the fluid feeding device is provided at a position where a force for inflow of the fluid can act due to the outflow of the fluid from the discharge port. In other words, the “position at which the force that the fluid tries to flow in can act” is a position that does not cancel the inertial force that causes the fluid to flow out from the discharge port.
(B) A position where only the force to flow in due to outflow from the discharge port acts, or
(B) Although the force of the fluid trying to flow out is exerted by the inertial force generated in the fluid in the storage chamber, the force of flowing in due to the outflow from the discharge port acts more than that force. Such positions are mentioned. As long as this condition is satisfied, the suction port may be provided on the inner side wall of the storage chamber or on the wall in the direction of the rotation axis of the rotor, but is preferably provided on the inner side wall.
The fluid feeding device of the present invention can be roughly divided into two modes depending on the positional relationship between the suction port and the discharge port.
One is the aspect (first aspect) of the above [5], for example, the apparatus shown in FIGS. That is, in the first aspect, when the suction port is assumed to be a linear or arc-shaped channel connecting the suction port and the discharge port, the channel is parallel to the wall in the direction of the rotation axis of the rotor. It is provided in such a position. For example, in FIGS. 1 and 2, a linear flow path 5 connecting the suction port 3 and the discharge port 4 can be assumed, and this flow path 5 is arranged in the direction of the rotation axis of the rotor in the housing chamber. It is parallel to a certain wall 2b.
In this aspect, in order to create a fluid flow in a certain direction by the rotation of the rotor, the rotation center 1a of the rotor is located on the central axis m of the flow path 5 as shown in FIG. It should not be a serious positional relationship. Therefore, in the first aspect, the positional relationship between the envisaged chamber and the flow path (and hence the suction port and the discharge port) is appropriately selected within a range as shown in FIGS. The 5D and 5E, it is considered that the upstream side of the flow of the portion where the inner side wall of the storage chamber is open with respect to the flow path is the suction port and the downstream side is the discharge port.
Although the suction port and the discharge port are provided at the same height, even if the linear or arc-shaped flow path connecting them is provided at a position that cannot be assumed. The discharge port is provided at a position where the fluid can flow out of the storage chamber from the discharge port due to the inertial force generated in the fluid in the storage chamber when the rotor is rotated, and the suction port is The inertial force generated in the fluid exerts a force that causes the fluid to flow out, but it is provided at a position where the force that flows in due to the outflow from the discharge port acts more than the force. As long as it is (for example, the case of FIG. 5 (f)), it can be included in the present invention.

The other one is the above-described aspect [8] (second aspect), and, for example, the apparatus shown in FIGS. 6A to 6C corresponds to this. That is, in the second aspect, the suction port is provided at a position different from the discharge port and at a position not including a portion surrounding the rotation circle drawn by the rotation of the rotor on the inner side wall. . When the rotor is rotated, the flow from the discharge port provided at the position including the portion surrounding the rotation circle of the inner side wall becomes dominant due to the inertial force. As a result, the fluid flows into the accommodation chamber from the suction port provided at a position not including the portion surrounding the rotation circle of the inner side wall as indicated by the arrow L1, and from the discharge port to the outside of the accommodation chamber as indicated by the arrow L2. To leak.
Even when the suction port is provided at a position different from the discharge port, and at a position including the portion surrounding the rotation circle drawn by the rotation of the rotor on the inner side wall, for example, As shown in FIG. 6 (d), the fluid flows out of the storage chamber from the discharge port due to the inertial force generated in the fluid in the storage chamber due to the rotation of the rotor clockwise, while the inertia generated in the fluid in the storage chamber. If the force by which the fluid tries to flow into the suction port from outside the storage chamber due to the outflow from the discharge port is larger than the force by which the fluid tries to flow out from the suction port, Since the fluid feeding action in which the fluid outside the storage chamber flows into the storage chamber from the suction port and flows out of the storage chamber from the discharge port when the is rotated, it can be included in the present invention.

  Hereinafter, the configuration of the fluid feeding device of the present invention will be described more specifically by dividing it into a first mode and a second mode.

  As described above, FIGS. 1 and 2 show an example of the first aspect of the fluid feeder of the present invention. In this aspect, when the suction port and the discharge port are assumed to be a linear or arcuate flow path connecting them, the positional relationship is such that the flow path is parallel to the wall in the rotation axis direction of the rotor. It is characterized by that. Therefore, when the rotor is accommodated so as to be horizontally rotatable, a horizontal flow path connecting the suction port and the discharge port is assumed. 1 and 2, a linear flow path 5 is assumed, but for example, an arc-shaped flow path as shown in FIG. 7 may be assumed. The assumed shape of the channel can be appropriately selected according to the shape of the culture channel connected to the suction port and the discharge port when the fluid feeder is used as a cell culture device.

  When the rotor is accommodated so as to be horizontally rotatable, the depth of the accommodation chamber is not limited as long as the rotor is rotatable, and the top plate of the accommodation chamber may be provided. However, if the upper and lower surfaces of the fluid are more excessively separated from the upper and lower surfaces of the rotor, an unintended flow such as a vortex occurs, and it becomes difficult to obtain a fluid feeding action by inertial force. From such a point, the gap d2 between the rotor and the ceiling of the storage chamber (or the fluid level) in FIG. 4B is a preferable value of about 0.1 mm to 10 mm, particularly 1 mm to 5 mm. . Further, the fluid level may be lower than the upper surface of the rotor.

  As described above, in a preferred embodiment of the present invention, the discharge port is provided at a position including at least a portion surrounding the rotation circle drawn by the rotation of the rotor on the inner side wall of the storage chamber. The depth d3 of the path can be appropriately selected between the gap d2 between the rotor and the ceiling of the storage chamber and the depth d1 of the storage chamber, but the fluid feeder is used as a part of the cell culture device. In general, the cells are held on the bottom surface of the culture channel connected to the suction port and the discharge port, so the depth of the channel is set within a range where sufficient oxygen supply to the cells is achieved. It is desirable to do. Specifically, in the example of FIG. 4B, when the thickness of the rotor is 3 mm and the depth d1 of the storage chamber is 4 mm, the depth of the flow path (therefore, the depth of the suction port and the discharge port). d3 is 1 to 4 mm, preferably 1.5 to 3 mm.

  The assumed width of the flow path can be appropriately selected according to the diameter of the rotating circle of the rotor, the type of cells to be cultured, and the like. For example, in the example of FIG. When the inner side wall has a diameter of 6.5 mm, it is about 0.5 to 5 mm, preferably 1 to 3 mm.

As described above, in the first aspect of the fluid feeder of the present invention, the rotor is accommodated so as to be parallel to the wall in the direction of the rotation axis of the rotor that connects the suction port and the discharge port. Since the flow path is assumed to be horizontal, the rotor is rotated when the rotation center 1a of the rotor is on the assumed flow path center axis 5a as shown in FIG. However, no fluid flows in a certain direction due to inertial force. Therefore, in this aspect, it is necessary that the central axis of the assumed flow path does not pass through the rotation center of the rotor.
As an embodiment satisfying such conditions, when the inner side walls of the storage chamber are present at both ends of the flow path (FIG. 5B; in this case, the suction port and the discharge port are clearly distinguished), The rotation circle drawn by the rotation of the rotor circumscribes the side surface of the flow path connecting the suction port and the discharge port, or crosses one side surface but does not cross the other side surface (see FIG. It depicts the case where the inner side wall is inscribed in the side surface of the flow path when the inner side wall of the storage chamber is assumed to extend into the flow path along the rotation circle drawn by the rotation of the rotor). The rotation circle drawn by the rotation of the rotor does not come into contact with the flow path connecting the suction port and the discharge port (FIG. 5E). 5 (c) to 5 (e), the suction port and the discharge port are not visually distinguishable. However, the side on which the fluid flows in the opening of the inner side wall of the storage chamber is the suction port, the fluid The side from which the gas flows out is regarded as the discharge port.
In the embodiment as shown in FIG. 5B, the inertial force due to the rotation of the rotor does not efficiently cause the fluid flow in a certain direction. In the embodiment as shown in FIG. If the outlet is far from the rotor, it may be difficult to obtain a sufficient fluid feeding action. Therefore, the positional relationship between the assumed flow path connecting the suction port and the discharge port and the storage chamber is such that the rotation circle drawn by the rotation of the rotor circumscribes the side surface of the flow path or one side surface. It is preferable to cross the other side but not the other side surface, and it is more preferable that the inner side wall is inscribed in the side surface of the flow path when it is assumed that the inner side wall of the storage chamber extends into the flow path along the rotation circle. .
In the embodiment as shown in FIG. 5 (e), the shortest distance between the opening of the inner side wall of the storage chamber and the rotation circle is preferably about 0 mm to the diameter of the rotor, and 0 mm to a quarter of the rotor diameter is more preferable. preferable.
The diameter of the rotation circle drawn by the rotor and the diameter of the inner side wall of the storage chamber can be appropriately selected according to the assumed width of the flow path connecting the suction port and the discharge port, for example, the width of the flow path Is 3 mm, the diameter of the rotating circle is about 4 to 10 mm, preferably 5 to 8 mm. As the diameter of the inner side wall, a value about 0.2 to 5 mm, preferably 0.5 to 2 mm larger than the diameter of the rotating circle can be selected.

Next, the configuration of the second aspect of the fluid feeder according to the present invention will be described.
FIGS. 6A to 6C are schematic views showing examples of the second mode of the fluid feeder. As described above, in this aspect, the suction port is provided at a position different from the discharge port and at a position not including a portion surrounding the rotation circle drawn by the rotation of the rotor on the inner side wall. It is characterized by that. As long as the suction port has the above positional relationship with respect to the rotation axis direction of the rotor, the suction port may be provided at any position on the inner side wall of the storage chamber with respect to the rotation surface direction of the rotor. 6 (a) to 6 (c) show, as a typical example, when the fluid feeder is viewed from above, (a) 180 °, (b) 90 °, (c) 0 ° with respect to the discharge port. Each of them is provided with a suction port so as to have a positional relationship of (360 °). In each figure, the upper part is a view of the apparatus as viewed from above, and the lower part is a cross-sectional view when the apparatus is cut in the lateral direction of the paper surface so as to pass through the rotation center of the rotor.
The suction port has a different positional relationship with the discharge port in the rotation axis direction of the rotor, and when the rotor is accommodated in the storage chamber so as to be horizontally rotatable, the suction port is provided at a position higher than the discharge port. Will be. The discharge port is provided at a position including at least a part of a portion surrounding the rotation circle drawn by the rotation of the rotor on the inner side wall, whereas the suction port is a portion of the inner side wall surrounding the rotation circle. When the rotor is rotated, the flow from the discharge port to the outside of the storage chamber is dominant due to the inertial force when the rotor is rotated. As a result, the fluid flows from the suction port into the storage chamber. Then, the fluid flows out from the discharge port to the outside of the storage chamber, and a fluid feeding action occurs.
The difference between the first mode and the second mode is that the suction port and the discharge port are not connected by a flow path parallel to the wall in the rotation axis direction of the storage chamber, but are in a different positional relationship. It is.

  On the other hand, in FIG. 6D, the vertical relationship between the suction port and the discharge port is reversed from that in the first mode. That is, the discharge port is provided at a position on the inner side wall that does not include a portion surrounding the rotation circle drawn by the rotation of the rotor, whereas the suction port is a portion of the inner side wall that surrounds the rotation circle. It is provided in the position including. However, in this case, since the flow path connected to the discharge port (that is, the flow path from the discharge port toward the outside of the storage chamber) is the tangential direction of the rotation circle, the inertia of the tangential direction of the fluid rotating in the storage chamber Force can be efficiently used as the discharge force, while the inertial force acting on the suction port is smaller than the force of fluid flowing from the outside of the storage chamber to the suction port due to the outflow from the discharge port. Therefore, as a result, the fluid flows from the suction port into the storage chamber and flows out from the discharge port to the outside of the storage chamber, thereby causing a fluid feeding action.

  The opening areas of the suction port and the discharge port in the second aspect vary depending on the depth of the storage chamber, the diameter of the rotation circle drawn by the rotor, and the like, for example, the diameter of the rotation circle is 6 mm, and the diameter of the inner sidewall of the storage chamber Is 6.5 mm and the depth of the storage chamber is 5 mm, the suction port width (the length of the rotor in the direction of the rotation surface) is about 1 to 5 mm, preferably 2 to 4 mm, and the height of the suction port (rotation) The length of the rotor in the rotation axis direction) is about 0.5 to 5 mm, preferably 1 to 3 mm, and the discharge port width (the length of the rotor in the rotation surface direction) is about 1 to 5 mm, preferably 2 to 4 mm. The height of the discharge port (the length of the rotor in the rotation axis direction) is about 0.5 to 5 mm, preferably 1 to 3 mm.

Next, a cell culture apparatus configured using the fluid feeder according to the present invention will be described.
For convenience, an example using the fluid feeding device of the first aspect will be described in detail, but the fluid feeding device of the second aspect or the other of the present invention shown in FIGS. 5 (f) and 6 (d) The fluid feeding device according to the aspect may be appropriately replaced.
As shown in FIGS. 8 and 9, the cell culture device is configured by connecting a fluid feeding device according to the present invention and a culture channel. A culture substrate can be disposed in the culture channel, and when cells of interest are seeded in the culture channel, the cells are retained in the culture substrate or on the surface thereof. The cell is cultured by being distributed. One end (first end) of the culture channel is at the suction port of the fluid feeder and the other end (second end) is at the discharge port so that the culture fluid flows through the culture channel. , Each connected.

An important feature of the cell culture device is that the fluid feeding device has a sufficiently simple structure and is miniaturized, so that the fluid feeding device and the culture channel are small in the culture solution in the culture tank. The circulation culture in which the culture solution is moved in a certain direction only in the culture tank without passing through the circulation path outside the culture tank is established.
Strictly speaking, the cell culture device has a stirrer main body driving device attached to the outside. However, when a magnetic stirrer is used, the rotor is not mechanically connected. It can be considered as only a circulating culture device.
Even if the stirrer body drive device is included, the entire device is compact, so the entire device including the stirrer body drive device should be placed as it is in the tanks of various devices such as carbon dioxide incubators and thermostats. Is also possible.
In particular, the one that inhales the culture solution horizontally and discharges it horizontally has a thin and compact structure. When this is combined with a horizontal culture channel, it becomes a very shallow dish like a petri dish or well. It becomes possible to circulate the culture solution horizontally in the culture tank. There is no mechanical connection with the outside world related to the driving of the fluid feed, and cell culture in two or three dimensions is performed only inside a thin, small-diameter petri dish.

Further, in the second mode and other modes of the fluid feeding device, cell culture that circulates in a shallow culture tank can be similarly performed by connecting a horizontal culture channel to the suction port.
Further, even in the first aspect, the second aspect, and other aspects of the fluid feeding device, depending on the rotational posture of the rotor and how to take the suction port and the discharge port, the vertical discharge and suction may be used. become. Therefore, a vertically elongated cell culture device such as a test tube or a graduated cylinder can be configured as a whole by using a culture channel / culture tank elongated in the vertical direction.
In any embodiment, since the culture tank itself may be an open container without a lid, it does not accumulate in the tank even if bubbles are generated.
In addition, it is a structure that can generate a flow of a culture solution containing nutrient factors and oxygen in a certain direction and always supply them to cells and tissues. In addition, depletion of essential culture components such as amino acids and oxygen can be suppressed, and long-term culture and cell function can be maintained.

The cross-sectional shape of the culture channel (the cross-sectional shape when cut perpendicular to the flow direction) is not particularly limited, and may be circular, square, or oval as long as it matches the inlet and outlet of the fluid feeder. Any of these may be used.
The culture channel may be a tubular channel, but in the case of a horizontal channel, the upper surface (ceiling portion) or the like may be open for reasons such as good oxygen supply.
When the example of a preferable dimension of the cross section when the cross-sectional shape is a square, a flow path width of 0.1 mm to 10 mm and a flow path depth of 0.1 mm to 10 mm are compact.

When constructing a cell culture device extending in the horizontal direction as shown in FIG. 8 and FIG. 9, if the storage chamber of the fluid feeder and the culture channel are integrally formed from one member, the configuration Becomes easier. Examples of such a member include an acrylic plate, stainless steel, polystyrene, polycarbonate, and hydroxyapatite, but an acrylic plate and polystyrene are more preferable from the viewpoints of ease of processing, durability, transparency, and easy observation. Can be used.
In addition, when the culture channel directly connected to the discharge port as shown in FIG. 10 is formed to open downward, the formed member is placed on the bottom surface of a petri dish, well plate, stainless steel plate, etc. A culture device can also be constructed by bonding using a potting agent.

When constructing a cell culture device extending in the horizontal direction as shown in FIGS. 8 to 10, in order to make use of the compactness of the fluid feeding device, the shape of the culture tank has an inner diameter (or one side) of 5 mm to 35 mm, A cylinder (quadrangular prism) shape having a depth of about 5 mm to 20 mm is exemplified.
What is necessary is just to set the liquid level height of a culture tank so that it may be 0 mm-3 mm, especially about 0 mm-1 mm lower than the storage chamber of a fluid feeder, and the upper surface (ceiling) of a culture flow path.

  One end (first end) of the culture channel is connected to the suction port of the fluid feeder, and the other end (second end) is connected to the discharge port. As shown in FIGS. 8 to 10, if the first end of the culture channel 5 is connected to the suction port of the fluid feeder and the second end is connected to the discharge port, and the rotor is rotated, the rotor is rotated. Due to the inertial force of the culture solution generated in the room, the culture solution flows into the culture channel from the discharge port, circulates through the culture channel, and flows again into the accommodation chamber from the suction port.

As the culture channel, a bent channel as shown in FIG. 8, an annular channel as shown in FIG. 9, a linear channel as shown in FIG. 10, and the like can be used in appropriate combination. By disposing the culture channel in the culture tank so as to extend substantially horizontally, a thin and compact cell culture device can be configured.
FIG. 8 is a combination of the first embodiment of the fluid feeder and the square bent culture channel 5. 8A is a view of the apparatus as viewed from above, FIG. 8B is a cross-sectional view taken along X1-Y1, and FIG. 8C is a cross-sectional view of the storage chamber.
FIG. 9 is a combination of the first embodiment of the fluid feeder and the annular culture channel 5. FIG. 9A is a view of the apparatus from above, and FIG. 8B is a cross-sectional view of the storage chamber. By making the culture channel annular, a smooth flow of the culture solution can be achieved without stagnation.
FIG. 10 shows a combination of (a) a hairpin-type bent channel 5 or (b) a straight channel 5 and a semi-annular channel 6 in the second mode of the fluid feeder. In each figure, the upper part is a view of the apparatus viewed from above, and the lower part is a cross-sectional view when the apparatus is cut along X2-Y2 and X3-Y3.

The culture substrate can be appropriately disposed in the culture channel. The culture substrate may be arranged in the entire range of the culture channel or may be arranged in a part. In the case of using the fluid feeding device of the second aspect, it is troublesome to arrange the culture substrate in the culture flow path portion directly connected to the discharge port, so that the culture medium is placed in the portion directly connected to the suction port whose upper surface is open. It is preferable to arrange the material. On the other hand, when the first aspect of the fluid feeder is used, since a horizontal culture channel having an open upper surface can be used, the culture substrate can be easily disposed in the entire culture channel.
The culture substrate is not particularly limited as long as it is a scaffold material suitable for various cells and tissues, and known materials can be used.
The culture substrate is arranged in the culture channel so that the culture solution can pass through the culture substrate (the cultured cells are contained inside or on the surface) during the culture. Just do it. The phrase “the culture solution passes through the culture substrate” means that the culture solution passes through the gap between the culture substrate and the flow path, and / or the culture solution passes through the culture substrate. In particular, a mode in which the culture substrate is configured as a porous substrate and the culture solution passes so as to contact the cells in every corner of the culture substrate is preferable.
A culture base material capable of three-dimensional culture is a porous base material because it is mainly a porous member.

The culture substrate that can be used in the present invention is not particularly limited, and may be a porous carrier capable of fixing cells and tissues on the contact surface, a hollow fiber, or the like. More specifically, cellulose, ceramics, urethane, polystyrene, metal, glass, polypropylene, collagen, fibronectin, polylysine, lamin, dextran and the like can be mentioned.
The cells to be cultured usually remain in the culture substrate (or the surface), and the culture solution passes around the cells.

The cells that can be cultured in the cell culture apparatus of the present invention are not particularly limited, and may be cells derived from any organism (prokaryotes, eukaryotes, etc.). Cell culture includes not only simple cell culture but also tissue and organ culture.
Prokaryotes include bacteria such as E. coli. Examples of eukaryotes include fungi (yeast (fission yeast, germinating yeast, etc.)), plants (monocotyledonous plants, dicotyledonous plants, etc.), animals (invertebrates, vertebrates, etc.) and the like. Examples of invertebrates include crustaceans (insects, etc.). Examples of vertebrates include eel eels, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals and the like.

  There is no limitation on the culture medium that can be used in the present invention, and a culture medium that can culture desired cells in combination with the above culture substrate can be appropriately selected. For example, when culturing mammalian cells using the cell culture apparatus of the present invention, culture media known per se, such as DMEM, EMEM, RPMI-1640, α-MEM, F-12, F-10, M- 199, HAM, etc. can be used. A known additive (serum, growth factor, antibiotic, buffer, reducing agent, etc.) may be appropriately added to the culture solution.

The use of the cell culture device according to the present invention is not limited, but for example, culture for the purpose of enhancing the functionality of cells, culture for the purpose of producing substances such as medical proteins, culture of embryonic stem cells, tissue culture, cells Culture for the purpose of morphological observation of tissues and tissues, culture for the purpose of testing / inspection, screening assay for anticancer substances and antioxidants, other enzyme reaction tests, and interaction of compounds including biomolecules such as nucleic acids and proteins It may be used for various purposes such as testing, substance purification, and compound synthesis.
Preferably, the cell culture device of the present invention cultivates various functional cells such as hepatocytes and kidney cells, and monitors the change in the drug concentration while adding and distributing the drug in the culture solution. It can be used as a drug metabolism simulator in the body.

  As shown in FIG. 11 (b), the unit shown in FIGS. 8 to 10 [fluid feeder and culture can be used if a device capable of simultaneously rotating a plurality of rotors on the stage of one stirrer body drive device S is used. A plurality of units (A1 to A6) having a configuration in which a flow path is connected are arranged on the stirrer main body driving device S, and the plurality of units A1 to A6 are operated by one common stirrer main body driving device. Therefore, even in a narrow space, it becomes possible to process various samples in parallel. FIG. 11B shows a configuration example in which 6-wells are used as each culture tank and are arranged on the stirrer main body driving device S.

  The present invention will be described more specifically with reference to the following examples. However, these are merely examples and do not limit the present invention.

Example 1 Manufacture of various culture apparatuses The apparatus (Type I) shown in FIG. 10 (a), the apparatus (Type II and Type III) shown in FIG. 10 (b), the apparatus (Type IV and Type V) shown in FIG. The apparatus (Type VI) shown in FIG. 9 was actually manufactured. Details of the material, shape (dimensions), etc. of each culture apparatus are as follows.
(Type I)
Channels with a depth of 2 mm, width of 2 mm, and length of 15 mm were provided on the upper and lower surfaces of a 5 mm thick acrylic disk. In addition, two holes penetrating the acrylic disk were provided so that the medium could circulate through the upper and lower flow paths using the rotation of the stirrer as a driving force. The diameter of the hole (accommodating chamber) into which the stirrer was placed was 6.7 mm, and the diameter of the other hole was 3 mm.
(Type II / Type III)
A semicircular channel with a depth of 2mm and a width of 3mm on the upper surface of a 5mm-thick acrylic disc, and a linear channel with a depth of 2mm and a width of 2mm on the lower surface of the acrylic disc (length: 15mm (Type II), 20mm (Type III)). In addition, two holes penetrating the acrylic disk were provided so that the medium could circulate through the upper and lower flow paths using the rotation of the stirrer as a driving force. The diameter of the hole (accommodating chamber) into which the stirrer was placed was 6.7 mm, and the diameter of the other hole was 4 mm.
(Type IV / Type V)
A square channel with a width of 3 mm was provided on the top surface of an acrylic disk having a thickness of 5 mm. The depth of the Type IV channel was 2 mm, and the depth of the Type V channel was 4 mm. The diameter of the hole (accommodation chamber) for containing the stirrer was 6.5 mm and the depth was 4 mm.
(Type VI)
An annular channel with a depth of 2 mm and a width of 3 mm was provided on the top surface of an acrylic disk having a thickness of 5 mm. The diameter of the hole (accommodation chamber) for containing the stirrer was 6.5 mm and the depth was 4 mm.

Example 2 Culture of Hepatocytes Using Various Culture Devices Hepatocytes were cultured using the various culture devices (Type I to Type VI) produced in Example 1.
(Type I)
Collagen was placed in the shape of the lower channel using a stainless steel template, and a rat hepatocyte suspension collected by a conventional method was added, followed by static culture for 4 hours in an open system. The template was removed, and a stirrer was placed in a storage chamber incorporating the acrylic cone formed as described above, and the stirrer was rotated clockwise at 100 rpm using a magnetic stirrer, and flow culture was performed for 3 days.
(Type II / Type III)
The acrylic disk molded as described above was adhered to a Petri dish. Collagen is placed on the bottom of the top channel as a culture substrate. After adding rat hepatocyte suspension collected by conventional methods and incubating for 4 hours, a stir bar is placed in the storage chamber and a magnetic stirrer is installed. The stirring bar was rotated clockwise at 100 rpm, and flow culture was performed for 3 days. As a control, the culture was carried out as it was without circulating the medium.
(Type IV / Type V)
Place the collagen as the culture substrate on the bottom of the acrylic disk shaped as described above, add the rat hepatocyte suspension collected by the usual method and incubate for 4 hours, then place the stir bar in the storage chamber. Then, the stirring bar was rotated clockwise at 100 rpm using a magnetic stirrer, and flow culture was performed for 3 days. As a control, the culture was carried out as it was without circulating the medium.
(Type VI)
Place collagen as a culture substrate on the bottom of the flow path of the acrylic disk molded as described above, place a cylindrical member in the accommodation chamber, dam the culture medium, and add the rat hepatocyte suspension collected by a conventional method. After stationary culture for 4 hours, the weir was removed, a stirrer was placed in the accommodation chamber, and the stirrer was rotated clockwise at 100 rpm using a magnetic stirrer, and flow culture was performed for 3 days. As a control, the culture was carried out as it was without circulating the medium.
Table 1 summarizes the channel depth, medium volume, cell seeding bottom area, and number of seeded cells of each culture apparatus.

Test Example 1 Performance of Various Culture Devices In the various culture systems of Example 2, albumin synthesis ability was measured on the first and third days of culture. The results are shown in FIGS. Type I had a relatively low albumin synthesis ability because the circulation speed was insufficient at the number of rotations used (FIG. 12).
In both Type II and Type III, the rate of albumin synthesis on the first day of culture exceeded the value in static culture in the flow culture (FIG. 13). However, on the third day of culture, the albumin synthesis rate decreased for both Type II and Type III. The reason for this is that when the culture device was affixed to the Petri dish using a potting agent, the coating was uneven, and the medium soaked between the reactor and the Petri dish, resulting in a decrease in the liquid level in the upper channel. It is possible that On the third day of culture, a state in which there was almost no medium in the flow path was observed.
In Type IV and Type V, both the first and third days of culture exceeded the results of static culture (FIG. 14). In particular, in Type IV, an extremely high value was obtained as compared with the albumin synthesis rate obtained so far by static culture. In addition, since the experimental operation was simple, it was expected to be applied to a distribution-type drug metabolism simulator. However, since it has a square channel, stagnation was observed at the four corners of the channel when a dye was added and the flow state was observed.
As with Type IV and Type V, Type VI exceeded the results of static culture on day 1 and day 3 (FIG. 15). The cell morphology on the second day of culture was also good (FIG. 16). Moreover, since the flow path was made into an annular shape, no stagnation was observed when the flow condition was observed with the addition of a dye, and a smooth flow of the culture medium became possible. Therefore, we tried to apply Type VI to a distribution-type drug metabolism simulator.

Test Example 2 Drug Metabolism Ability in Flow Culture Using Type VI Culture Device In the same manner as in Example 2, hepatocytes were cultured in a Type VI culture device. Flow culture or stationary culture was performed by adding 0.25 mM lidocaine to the medium, and the lidocaine concentration was measured 6 hours and 24 hours later to calculate the clearance value. The results are shown in Table 2. The predicted value of biological clearance obtained by flow culture was about 30% higher than that obtained by static culture.

  As described above, the fluid feeder according to the present invention has a simple configuration in which a rotor and a flow path are combined, and thus reflux culture can be performed in a small culture tank such as a well plate. Therefore, it is possible to process a large number of samples at a time, and it can be used for high-throughput cell function evaluation and the like.

It is a schematic diagram for demonstrating an example of the structure of the fluid feeder by this invention, and demonstrating the principle of the said fluid feeder. It is a figure which shows the XY cross section of the apparatus shown in FIG. It is a figure for demonstrating the clearance gap between a rotation circle and the internal wall surface of a storage chamber in the fluid feeder by this invention. It is a figure for demonstrating the position of the discharge outlet, the clearance gap between a rotor, and the ceiling of a storage chamber etc. in the fluid feeder by this invention. (A)-(e) In the fluid feeder by this invention, it is a figure which illustrates the positional relationship of the rotation center of a rotor, and the centerline of a flow path. (F) It is a figure which shows the positional relationship of the suction inlet and discharge outlet in the fluid feeder of another aspect of this invention. It is a figure which illustrates the positional relationship of the suction inlet and discharge outlet in the fluid feeder by this invention. It is a figure which shows an example of the shape of the flow path in the fluid feeder by this invention. It is a schematic diagram which shows an example of a structure of the cell culture apparatus by this invention. It is a schematic diagram which shows an example of a structure of the cell culture apparatus by this invention. In the cell culture apparatus by this invention, it is a schematic diagram which shows the structural example in case the culture flow path directly connected to a discharge outlet is open | released below. FIG. 10A is a top view of the device and its X1-Y1 cross section, and FIG. 10B is a top view of another example of the device and its X2-Y2 cross section. In the cell culture device by this invention, it is a figure which shows the aspect made into the structure which can rotate a some rotor simultaneously on the stage of one stirrer main body drive device. It is a figure which shows the time-dependent change (n = 2) of the albumin synthesis | combination rate per cell seed | inoculation area in the flow culture using a Type I culture apparatus. It is a figure which shows a time-dependent change (n = 2) of the albumin synthesis | combination rate per cell seed | inoculation area in distribution culture and stationary culture. (a) Type II, (b) Type III It is a figure which shows a time-dependent change (n = 2) of the albumin synthesis | combination rate per cell seed | inoculation area in distribution culture and stationary culture. ■: Type IV flow culture, △: Type V flow culture, ●: Type IV static culture, ▽: Type V static culture It is a figure which shows the time-dependent change (n = 2) of the albumin synthesis | combination rate per cell seeding | spread area | region in distribution culture and stationary culture using a Type VI culture apparatus. It is a photograph which shows the cell form of the culture | cultivation 2nd day in distribution culture using a Type VI culture apparatus, and stationary culture. A: Type VI flow culture, B: Type VI static culture, C: static culture using 12 well dishes

Explanation of symbols

1 Rotor 2 Storage chamber 3 Suction port 4 Discharge port

Claims (15)

The stirrer rotor has a storage chamber in which the rotor is rotatably stored, and the storage chamber is provided with a suction port and a discharge port for allowing fluid to enter and exit,
The discharge port is provided on the inner side wall of the storage chamber at a position where the fluid can flow out of the storage chamber from the discharge port due to inertial force generated in the fluid in the storage chamber when the rotor is rotated.
When the rotor is rotated, the suction port
(B) A position where only the force to flow in due to outflow from the discharge port acts, or
(B) Although the force of the fluid trying to flow out is exerted by the inertial force generated in the fluid in the storage chamber, the force of flowing in due to the outflow from the discharge port acts more than that force. It is provided in such a position,
Thereby, when the rotor is rotated, a fluid feeding action in which the fluid outside the storage chamber flows into the storage chamber from the suction port and flows out of the storage chamber from the discharge port,
Fluid feeder.   The fluid feeding device according to claim 1, wherein the discharge port is provided at a position including at least a portion surrounding a rotation circle drawn by rotation of the rotor on the inner side wall of the storage chamber.   The fluid feeding device according to claim 1, wherein the discharge port is provided at a position not including a portion surrounding a rotation circle drawn by rotation of the rotor on the inner side wall of the storage chamber.   The fluid feeder according to claim 1, wherein the suction port is provided on an inner side wall of the storage chamber.   When the suction port is assumed to be a linear or arc-shaped flow path connecting the suction port and the discharge port, the flow path is provided at a position parallel to the wall in the direction of the rotation axis of the rotor. The fluid feeder according to claim 4, wherein the central axis of the flow path does not pass through the rotation center of the rotor.   The fluid feeding device according to claim 5, wherein the rotation circle drawn by the rotation circle of the rotor circumscribes the side surface of the flow path connecting the suction port and the discharge port, or crosses one side surface but does not cross the other side surface. .   The fluid feed according to claim 6, wherein the inner side wall is inscribed in the side surface of the flow path when it is assumed that the inner side wall of the storage chamber extends into the flow path along a rotation circle drawn by rotation of the rotor. apparatus.   The fluid feed according to claim 2, wherein the suction port is provided at a position different from the discharge port and at a position on the inner side wall that does not include a portion surrounding a rotation circle drawn by rotation of the rotor. apparatus.   The fluid feeding device according to claim 1, wherein the stirrer rotor is housed in the housing chamber so as to be horizontally rotatable.   The fluid feeding device according to any one of claims 1 to 9, wherein the stirrer is a magnetic stirrer, and the rotor is rotated by the action of magnetism emitted from a stirrer main body driving device disposed outside the storage chamber. . A cell culture device comprising the fluid feeder according to any one of claims 1 to 10 and a culture channel provided outside a storage chamber of the fluid feeder,
One end of the culture channel is connected to the inlet of the fluid feeder, and the other end is connected to the outlet of the fluid feeder,
A cell culture device, wherein the culture solution is configured to pass through a culture channel by a fluid feed action of the fluid feed device. The stirrer of the fluid feeding device is a magnetic stirrer, and the rotor rotates horizontally by the action of magnetism emitted from the stirrer main body driving device arranged outside the storage chamber,
The stirrer main body driving device is configured to be capable of rotating a plurality of rotors by one unit.
A plurality of units including a configuration in which the fluid feeding device and the culture channel are connected are arranged on the stirrer body driving device, and the plurality of units are operated by one stirrer body driving device. The cell culture device according to claim 11.   The cell culture device according to claim 11 or 12, which is a drug metabolism simulator for cells.   A culture substrate is disposed in the culture channel of the cell culture device according to claim 11 or 12, a culture medium is added to the culture channel and the storage chamber of the fluid feeding device, the cells are seeded, and the cells are transformed into the culture medium. A cell culturing method, wherein the medium is circulated in a fixed direction in the culture channel by rotating the stirrer rotor of the fluid feeder after being held in the material or on the surface thereof.   A culture substrate is disposed in the culture channel of the cell culture device according to claim 11 or 12, a culture medium is added to the culture channel and the storage chamber of the fluid feeding device, the cells are seeded, and the cells are transformed into the culture medium. The drug metabolism of the cell, which comprises circulating the medium containing the drug by rotating the stirrer rotator of the fluid feeder and measuring the change in the drug concentration after being held in or on the surface of the material Simulation method.