JP3024151B2 - Cell separation device in cell culture device - Google Patents

Abstract

PURPOSE:To obtain the title device eliminating complicated maintenance, by making constitution of upward moving cells in a liquid along an inner wall face of rotary tank having diameter lessening downward set laid in a storage tank by centrifugal force through rotation of the rotary tank and sending to the outer peripheral side. CONSTITUTION:A liquid medium and desired cells are fed to a culture tank 11 and a rotary tank 14 is revolved by rotation of a magnet 31 to stir the liquid medium. Then, the cells subjected to centrifugal force are moved outside while moving the cells upward in the tank 14. The cells moved upward and a culture solution are delivered from an outlet hole 44 to the outside of the tank 14. Then the delivered cells are transferred downward between the tank 14 and the tank 11 and resent from an open hole 41 at the lower end of the tank 14 to the tank 14. In this way, the cells in the tank 11 are circulated in the vertical direction like the broken line. In the circulation, since the cells moved upward are delivered from the outlet hole 44 to the outside, only the culture substance separated from the cells are left at the upper central part of the tank 14. Then the culture solution is recovered in a recovery medium tank 13.

Description

Description: TECHNICAL FIELD The present invention relates to a cell separation device, and particularly to a cell separation device provided in a cell culture device.

[Conventional technology and its problems]

Cell culture devices for collecting monoclonal antibodies, collecting large amounts of nucleic acids and intracellular components, studying cell culture conditions, and collecting large amounts of cell products are already known. This type of cell culture apparatus includes a culture tank in which culture conditions are controlled, a fresh medium tank containing a fresh liquid medium, a collection medium tank for storing used liquid medium, a culture tank and a collection medium. The apparatus includes a filter (cell separation device) arranged between the tank and the tank, and a circulation path for returning the cells separated by the filter to the culture tank.

In this cell culture device, a fresh liquid medium is supplied from the fresh medium tank to the culture tank as needed using a pump mechanism. In addition, a liquid medium containing cells in the culture tank is introduced into the filter by a pump mechanism to separate the cells from a liquid medium containing waste products and useful products. The separated cells are returned to the culture tank through the circulation path. On the other hand, the remaining liquid medium is collected in the collection medium tank.

As the above-mentioned filter, a filter using a porous ceramic cylinder is generally used. In this case, the ceramic cylinder has such small holes that cells cannot pass through, and the liquid medium and the cells are separated by passing only the used liquid medium.

However, in this configuration, the ceramic cylinder is likely to be clogged, and the cylinder must be replaced frequently. For this reason, in a device using a ceramic cylinder, replacement is troublesome.

Therefore, a cell separation device 1 as shown in FIG. 4 is known as an alternative to the filter. The cell separation device 1 is a generally conical member that opens upward. At the upper end of the cell separation device 1, a culture medium outlet hole 2 connected to the collection medium tank is provided. In addition, a culture medium inlet 3 for introducing a liquid medium containing cells is provided at the lower end of the cell separation device 1.

In the cell separation device 1, a liquid medium containing cells is introduced from the medium inlet 3. Since the specific gravity of the cells is higher than that of the liquid medium, the cells do not rise so much in the cell separation device 1, and ideally, only the liquid medium is supplied to the medium inlet 2.
From the recovery medium tank.

However, in the cell separation device 1 shown in FIG. 4, when the flow rate in the device is slightly increased, the cells are easily discharged from the medium inlet 2 to the outside. On the other hand, since the culture medium flow rate is large near the culture medium inlet 3, the cells once introduced into the apparatus hardly fall down from the culture medium insertion port 3. Therefore, if used for a long period of time, the cells will be filled in the cell separation device 1. As for the cells trapped in the cell separation device 1, it becomes difficult to control the oxygen concentration, pH and temperature,
Cell culture cannot be performed efficiently.

The object of the present invention is to eliminate the need for frequent maintenance,
Moreover, it is an object of the present invention to provide a cell separation device in which a large amount of cells are not trapped inside.

[Means for solving the problem]

A cell separation device according to the present invention is a device provided in a cell culture device for separating cells from a liquid containing cells. This device includes a storage tank, a rotary tank, and a liquid outlet.

The storage tank stores a liquid. The rotary tank is rotatably provided in the storage tank, has an inlet formed in the lower part and an outlet formed in the upper part, and reduces the diameter as it goes downward, to separate cells from the liquid. belongs to. The liquid outlet is provided at the upper center of the rotary tank, and is for discharging the liquid from which the cells have been separated.

[Action]

In the cell separation device according to the present invention, first, a liquid containing cells is stored in the storage tank and the rotary tank. Next, the rotating tank is rotated. Then, the liquid containing cells rotates inside the rotary tank, and centrifugal force acts on the cells. The cells that have been subjected to the centrifugal force move upward along the inner wall surface of the rotating tank, and are led out from the outlet to the outer peripheral side. Thereby, the cells are separated from the liquid inside the rotating tank.

On the other hand, the liquid from which the cells have been separated is led out of the device from a liquid outlet provided in the upper center of the rotary tank.

In this case, since it is not necessary to use a porous ceramic member or the like, frequent maintenance such as replacement of the member is not required. Further, since the cells separated from the liquid in the rotary tank are led out from the outlet, there is no problem that a large amount of cells are trapped inside the device.

〔Example〕

FIG. 1 shows a cell culture apparatus employing one embodiment of the present invention.

In FIG. 1, a cell culture device includes a culture tank 11 as a storage tank, a fresh medium tank 12 for supplying a fresh liquid medium to the culture tank 11, and a liquid medium containing waste products from the culture tank 11. It mainly has a collection medium tank 13 for collection and a rotary tank 14 provided inside the culture tank 11.

The culture tank 11 is a closed container whose upper end is closed by a lid 15. In the culture tank 11, a culture solution discharge pipe 16 provided through the lid 15 and a culture solution introduction pipe 18 are arranged. One end of a tube 20 having an ironing pump 19 on the way is connected to the upper end of the culture solution discharge pipe 16. One end of a tube 23 having an ironing pump 22 on the way is connected to the upper end of the culture solution introduction pipe 18.

A rotating tank 14 is rotatably provided in the culture tank 11. The rotary tank 14 is formed using a material that can be autoclaved, for example, glass or polycarbonate. As shown in FIG. 2, the rotary tub 14 includes a rotary tub main body 40 whose diameter decreases as going downward, a support portion 43 provided at an upper end of the rotary tub main body 40 and protruding upward, And a magnetic member 47 provided at the lower part. Rotating tank body
A plurality of outlet holes 44 are formed in the upper part of the side wall of the forty. Also, as shown in FIG.
On fins 45, fins 46 extending vertically (in a direction perpendicular to the plane of FIG. 3) are provided. These fins 46 are formed integrally with the rotary tank body 40. The support portion 43 has an opening 42 communicating with the inside of the rotary tank body 40.
In the culture tank 11, the culture solution discharge pipe 16
The bearing 26 (FIG. 1) is mounted between the support portion 43 and the pipe 16. This allows
The rotating tank 14 is rotatable around a culture solution discharge pipe 16.

Below the culture tank 11, a magnet 31 is rotatably provided. The magnet 31 is opposed to the magnetic member 47 of the rotary tub 14. The rotation of the magnet 31 causes the magnetic member 47 to receive its magnetic force, and the rotary tub 14 is rotated. The shaft 32 is fixed to the magnet 31. The motor 32 is connected to the shaft 32 via a pulley 33, a belt 34, and a pulley 35.
Are connected. The motor 36 is electrically connected to a control unit (not shown).

Further, a temperature sensor (not shown), p
An H sensor, a DO sensor, and a level sensor are arranged, and these sensors are electrically connected to a control unit (not shown). Further, an oxygen supply source, a carbon dioxide supply source, and a nitrogen gas supply source (not shown) are connected to the culture tank 11.
The control unit controls the amount of substance supplied from those supply sources based on the detection results of the above-described sensors. The culture tank 11 is also provided with a heater (not shown), and this heater is also controlled by the control unit.

In the fresh medium tank 12, a pipe 24 is arranged. The other end of the tube 23 is connected to the upper end of the pipe 24.
In this fresh medium tank 12, a fresh liquid medium can be stored. A pipe 25 is disposed in the collection medium tank 13. The other end of the tube 20 is connected to the upper end of the pipe 25. In the collection medium tank 13, the old culture solution used in the culture tank 11 is collected.

 Next, the operation of the above embodiment will be described.

First, the rotating tank 14 is set in the culture tank 11. And
A liquid medium and desired cells are put into the culture tank 11, the lid 15 is closed, and the culture tank 11 is sealed. Note that microcarriers are also placed in the culture tank 11 as necessary. Next, the motor 36 is driven to rotate the magnet 31 via the pulley 35, the belt 34, and the pulley 33. Due to the rotation of the magnet 31, the magnetic member 47, that is, the rotating tank 14, starts rotating, and the liquid medium is stirred. Here, centrifugal force acts on the cells in the culture solution when the culture solution inside the rotation tank 14 is rotated by the rotation of the rotation bath 14. The cells that have been subjected to the centrifugal force move outward in the rotating tank 14 as shown by the dashed arrows in FIG. The cells that have moved upwards are
From 44, it is led out of the rotary tank 14 together with the culture solution. The derived cells move downward between the rotating tank 14 and the culture tank 11 and are introduced again into the rotating tank 14 through an opening 41 (FIG. 3) at the lower end of the rotating tank 14. Thus, the cells in the culture tank 11 are
It circulates up and down as indicated by the dashed arrow in the figure. The temperature, pH, dissolved oxygen concentration and the like are controlled by a control device (not shown) while performing the stirring accompanied by the vertical movement, and the cells are cultured in the culture tank 11. When necessary, the ironing pump 22 is operated to introduce the fresh medium in the fresh medium tank 12 into the culture tank 11.

On the other hand, when discharging the old culture solution in the culture tank 11, the separation function of the rotary tank 14 is used. That is, when the culture solution inside the rotation tank 14 is rotated by the rotation of the rotation tank 14, centrifugal force acts on the cells in the culture solution, so that the cells having a higher specific gravity than the culture solution move upward in the rotation tank 14. And move outward. Then, the cells that have moved upward are led out of the rotating tank 14 through the outlet hole 44, so that the rotating tank
In the upper center of 14, only the culture solution from which the cells were separated remains.

Next, by operating the ironing pump 19, the culture solution from which the cells have been separated is collected in the collection medium tank 13 through the culture solution discharge pipe 16 and the tube 20. In this case, the collection amount of the liquid medium is controlled by the transfer amount of the ironing pump 19. In other words, by controlling the ironing pump 19, the collection amount of the liquid medium in the collection medium tank 13 can be arbitrarily set.

In this case, since the cells are constantly circulating vertically in the culture tank 11, the separated cells are not trapped in the rotating tank 14. Also, this rotating tank
In 14, no porous filter is used, so that clogging does not occur and frequent maintenance such as filter replacement is not required.

[Other embodiments]

(A) In the above embodiment, the magnet 31 was used, and the culture tank was used.
Although the rotary tank 14 is rotated from the outside of 11, the application of the present invention is not limited to this. For example, a configuration may be adopted in which a drive unit including a motor is disposed above the culture tank 11 and the rotary tank 14 is directly rotated while being sealed with an oil seal or the like.

(B) Rotating tank for more complete cell separation
A filter device using a porous ceramic may be arranged between 14 and the ironing pump 19 (for example, at the position of A).

In this case, it is also necessary to provide a flow path for returning the cells separated by the filter device to the culture tank 11. In this configuration, since most of the cells have already been separated by the rotary tank 14, the filter device arranged at the position A is hardly clogged, and frequent maintenance is unnecessary.

(C) In the above-described embodiment, the stirring device is not particularly provided, and the stirring is simultaneously performed by the fins 46 inside the rotating tank 40. However, the stirring is performed separately from the rotating tank 14 in the culture tank 11. A device may be provided.

〔The invention's effect〕

According to the cell separation device of the present invention, since the above-described rotary tank is provided in the storage tank, the problem that a large amount of cells are trapped in the cell separation device is solved.
Also, frequent maintenance is not required.

[Brief description of the drawings]

FIG. 1 is a schematic partial cross-sectional view of a cell culture apparatus employing one embodiment of the present invention, FIG. 2 is a perspective view of a rotary tank, and FIG.
FIG. 4 is a schematic vertical sectional side view of a conventional example. 11 ... Culture tank, 14 ... Rotating tank, 16 ... Culture liquid discharge pipe, 26 ... Bearing, 30 ... Rotary driving device, 41 ... Inlet, 42 ... Opening, 44 ... Outflow hole.

Claims (1)

(57) [Claims] 1. A cell separation device provided in a cell culture device for separating said cells from a liquid containing cells, comprising: a storage tank in which said liquid is stored; and a rotatably provided in said storage tank. A rotating tank for separating cells from the liquid, which has an inlet formed at a lower part and an outlet formed at an upper part, and is reduced in diameter as it goes downward, and is provided at a central upper part of the rotating tank. And a liquid deriving unit for deriving the liquid from which the cells have been separated.