JP2739066B2 - Cell culture device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention is to supply a gas such as oxygen necessary for culturing cells into a culture solution (medium) in a culture tank and stir the culture solution. The present invention relates to a cell culture apparatus for performing tissue culture while dispersing cells in a culture solution, and particularly to a cell culture apparatus suitable for culturing animal and plant cells.

[Conventional technology]

As a cell culture device, a device that supplies gas such as oxygen necessary for culturing cells into a culture solution and stirs the culture solution to disperse the cells in the culture solution to culture the cells is known. . In order to improve the growth rate and culture efficiency of cells with this type of cell culture device, it is necessary to uniformly disperse the cells in the culture solution and supply nutrients, oxygen, and the like to the cells sufficiently. For that purpose, it is necessary to supply sufficient oxygen to the culture solution and to agitate the culture solution appropriately.

However, animal cells are larger than other microorganisms and the like, and the cell membrane surrounding the cells is fragile and easily broken, so it is necessary to agitate very slowly. There was a risk of damage. In the case of culturing cells supported on a fine particle carrier (microcarrier), the cells may fall off from the carrier due to agitation due to agitation or rising bubbles.

Therefore, in order to solve this, conventionally, a large number of stirring blades are provided, the stirring blades are made to move in a complicated manner, or a stirring device having a special shape such as a stirrer disclosed in JP-A-60-160882. The culture solution was stirred using a vessel. Also shown in the method and apparatus for bubble-free gassing of a tissue culture growth medium in a liquid or the like of JP-A-61-100190,
A gas is supplied without generating bubbles in the culture solution, and the membrane sheet is flexibly immersed in the culture solution and moved so that the gas is introduced and the liquid is stirred simultaneously. ing.

[Problems to be solved by the invention]

However, in order to sufficiently supply the gas such as oxygen and agitate the culture solution with these devices, a complicated device configuration is required and the device becomes expensive, and furthermore, the production and maintenance of the device require time and effort. I was

Therefore, an object of the present invention is to provide a cell culture apparatus that can supply a gas such as oxygen with a simple configuration and that can effectively stir a culture solution.

[Means for solving the problem]

In order to achieve the above object, the present invention provides a gas such as oxygen necessary for culturing cells in a culture solution, and agitates the culture solution to disperse the cells in the culture solution. In a cell culture device for performing tissue culture, the gas is supplied by a thin tube made of a gas-permeable polymer material, and the thin tube is swirled or assembled to form a cylindrical gas supply body having both ends opened. A cell culture device, wherein the gas supply body is disposed at the center of the culture solution, with the opening of the gas supply body arranged vertically, further stirring the culture solution.
Performing with a stirring blade disposed on the central axis of the cylindrical gas supply body, and a thin tube made of the polymer material,
This includes supplying oxygen produced by an oxygen producing apparatus such as a pressure fluctuation type oxygen producing apparatus.

[Action]

The supplied gas such as oxygen permeates through the membrane of the capillary made of a gas-permeable polymer material and dissolves in the culture solution without bubbles. And a cylindrical gas supply body is formed with this thin tube,
By arranging the culture solution with the top and bottom open at the center, the function as a draft tube is generated, and the stirring efficiency is improved.

At this time, the stirring efficiency of the culture solution can be further improved by performing stirring of the culture solution using a stirring blade disposed on the center axis of the cylindrical gas supply body. Further, by supplying gas having a high concentration of oxygen in the thin tube, sufficient oxygen can be dissolved in the culture solution.

〔Example〕

 Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

The cell culture device 1 removes a supply unit 2 having a culture solution supply system L for supplying a culture solution and a gas supply system G for supplying oxygen and the like, a culture unit 3 for culturing cells, waste products, and the like. The filter unit 4 is configured. Culture part 3
Is a culture tank 5, a stirring blade 6 disposed at an upper part in the culture tank 5, and a gas supply body 7 provided at a central part in the culture tank 5.
Further, a dissolved oxygen sensor 8, a pH sensor 9 and a level sensor 10 for various controls are provided in the culture tank 5.
Is provided.

The supply unit 2 includes a culture solution supply system L including a culture solution tank 11 and a culture solution pump 12 for supplying a fresh culture solution to the culture tank 5, and oxygen gas (O ₂ ) and nitrogen gas in the culture solution. (N ₂ ), air (air), and a gas supply system G for supplying carbon dioxide (CO ₂ ) for pH adjustment.
The culture solution and various gases are supplied depending on the situation.

The gas supply system G adjusts the amount of supply of various gases by adjusting the opening of the valve 13 provided in each circuit. The oxygen gas circuit Go and the carbon dioxide gas circuit Gc have controllers 14a respectively. , 15a and timers 14b, 15b are provided.

The replenishment circuit 14 of the oxygen gas circuit Go
a is connected to the dissolved oxygen sensor 8, and when the amount of dissolved oxygen in the culture solution detected by the dissolved oxygen sensor 8 falls below a predetermined amount, the controller 14a operates and the timer 14b
The valve 14c is opened for a predetermined period of time, and oxygen is supplied into the oxygen gas circuit Go. Similarly, the replenishment circuit 15 of the carbon dioxide circuit Gc
The controller 15a is connected to the pH sensor 9, and when the pH of the culture solution detected by the pH sensor 9 becomes high, the controller 15a operates, and the controller 15a operates the valve 15c for a predetermined time set in the timer 15b. Open to replenish carbon dioxide in the carbon dioxide circuit Gc.

As a source of the oxygen gas, an oxygen production device 16a is used, and an oxygen gas richer than the atmosphere (for example, O ₂ 90% to 93%,
It is formed so as to supply a gas) the balance being N _2. As the oxygen producing device 16a, for example, a pressure fluctuation type oxygen producing device using an adsorbent for selectively adsorbing nitrogen gas or the like is used, and continuously separates and produces oxygen in the air and supplies it. Further, as for the nitrogen gas, a gas having a high nitrogen content (for example, N ₂ 99%, O ₂ O 1) can be obtained by using the nitrogen production device 16b using an adsorbent for oxygen gas similarly to the oxygen production device 16a.
%), And the proportions of the various components can be adjusted to an optimum state.

The various gases are supplied into the culture solution via a gas supply 7 disposed in the culture tank 5. The gas supply 7 is formed by a thin tube 7a made of a gas-permeable polymer material, and is shown in, for example, a gas exchange capillary assembly and a gas exchange device disclosed in JP-A-56-51210. It is formed of a gas-permeable silicon rubber or the like, and can dissolve the various gases described above without foaming in the culture solution.

This makes it possible to supply gas such as oxygen without generating air bubbles in the culture solution, and particularly when culturing animal cells which are susceptible to damage as described above, or when cells are supported on a fine particle carrier (microcarrier) and cultured. In addition, the cells are not damaged or dropped from the carrier.

As shown in FIG. 2, the gas supply body 7 spirally turns the thin tube 7a or forms a cylindrical shape having both ends opened by appropriately gathering the thin tube 7a. ,
The opening is arranged up and down. As described above, by disposing the gas supply body 7 that supplies various gases,
The gas supply 7 acts as a draft tube when the liquid is stirred, and the upward flow U and the downward flow D of the culture solution in the culture tank 5 can be separated into the inner and outer peripheries of the gas supply 7. Thereby, a smooth circulation flow can be formed in the culture solution in the culture tank 5, and the state of dispersion of the cells in the culture solution can be improved.

On the other hand, the stirring blade 6 provided at the upper part in the culture tank 5
A boss member 18 disposed on the central axis of the gas supply body 7 and fixed to the drive shaft 17; and a plurality of plate-shaped stirring blades 19 provided on the outer peripheral portion of the lower surface of the boss member 18. The stirring blade 19
And a ring 20 for connecting the lower part of the outer edge of the ring. The stirring blades 19 are arranged in the direction of the normal to a circle centered on the drive shaft 17, and form a space at the rotation center of the stirring blade 6 inside each stirring blade 19.

The stirring blade 6 formed as described above rotates when the drive shaft 17 is rotationally driven by the drive super motor 17a,
The culture solution at the center of the stirring blade 6 is caused to flow substantially horizontally toward the wall of the culture tank 5 by the action of the centrifugal force accompanying the rotation of the stirring blade 19. When the culture solution reaches the wall surface of the culture tank 5, it flows between the gas supply body 7 and the wall surface toward the bottom of the culture tank 5. On the other hand, the culture solution inside the ring 20 rises to the center of the stirring blade 6 with the flow of the culture solution to the wall surface side,
As described above, the gas sequentially flows to the outer peripheral side of the stirring blade 6. That is, the culture solution on the inner peripheral side of the gas supply 7 rises with the rotation of the stirring blade 6 and is sucked into the center of the stirring blade 6.

In this way, the stirring blade 6 is connected to the gas supply 7 above the culture tank 5.
The culture solution in the culture tank 5 is rotated downward by rotating the gas supply body 7 toward the bottom of the culture tank 7 on the outer peripheral side of the gas supply body 7 and by the upward flow on the inner circumference side of the gas supply body 7. U and a circulating flow flowing evenly in the culture tank 5. Therefore, the gas supply 7 and the culture solution can be brought into sufficient contact,
The gas supply efficiency can be improved, the cells can be uniformly dispersed in the culture solution, and sufficient nutrients and oxygen can be supplied to the cells.

Further, by forming the stirring blade 6 as described above and disposing it above the culture tank 5, it is possible to generate the above-mentioned flow of the culture solution even at a very slow rotation speed of about 10 rotations per minute. Therefore, the cells can be uniformly dispersed in the culture solution without damaging the cells or falling off the carrier, and the cells can be cultured efficiently.

The filter unit 4 removes products and waste products generated during the cell culture process together with a part of the culture solution, and keeps the culture solution in the culture tank 5 in a predetermined state.
And culture medium circulation pump 22, extraction pump 23 and collection tank 2
The controller 25 connected to the level sensor 10 provided in the culture tank 5 controls the operation of the extraction pump 23 to automatically perform the regeneration operation of the filter 21. .

A predetermined amount of the culture solution in the culture tank 5 is supplied to the culture solution circulation pump 22.
And is introduced into the lower part of the outer chamber 21a of the filter 21 to remove products and wastes, and circulates in a path returning from the upper part of the outer chamber 21a to the culture tank 5. The drawing pump
Usually, 23 sucks the culture solution in the inner chamber 21b of the filter 21 together with the products and wastes and sends it out to the collection tank 24. The controller 23 according to the amount of the culture solution in the culture tank 5
When the operation of the filter 25 is performed, the drawing pump 23 reverses to reverse the culture solution from the inner chamber 21b of the filter 21 to the outer chamber 21a, thereby regenerating the filter 21.

By configuring the cell culture apparatus 1 in this way, fresh culture medium and necessary oxygen and the like can be always supplied into the culture tank 5 and culture medium containing products and waste products can be withdrawn. it can.

Therefore, by the action of the gas supply body 7 and the stirring blade 6,
Cells that are uniformly dispersed in the culture solution can always be provided with nutrients such as fresh culture solution and oxygen, and products and waste products can be continuously removed, thus always forming an optimal culture environment. Therefore, the growth rate and culture efficiency of the cells can be improved.

The configuration of the present invention is not limited to the above embodiment.For example, a stirring blade having another shape may be used as the stirring blade, or a plurality of stirring blades may be appropriately provided on the inner and outer circumferences of the cylindrical gas supply body. Alternatively, the culture solution can be agitated using the flow of the culture solution from the supply unit or the filter unit. Also, the supply of oxygen can be a supply of only air or a supply using an oxygen cylinder.

〔The invention's effect〕

The present invention, as described above, while performing the supply of gas in a thin tube made of a gas-permeable polymer material, forming a cylindrical gas supply body having both ends opened by swirling or collecting this, In the center of the culture solution, the opening is arranged up and down, so that gas such as oxygen can be supplied without generating bubbles in the culture solution, and the stirring efficiency of the culture solution is improved to improve the culture efficiency. Can be improved. Furthermore, since it is only necessary to form a thin tube made of a gas-permeable polymer material into a cylindrical shape with both ends open, it can be easily manufactured, the structure of the culture tank can be simplified, and the cell culture device can be manufactured at low cost. In addition to being able to be manufactured, maintenance such as cleaning and replacement becomes easy, and handling is improved.

Further, by stirring the culture solution with a stirring blade arranged on the central axis of the cylindrical thin tube, a circulating flow of the culture solution flowing evenly in the culture tank is generated, and the cells are uniformly dispersed in the culture solution. , The cells can be provided with sufficient nutrients, and the culture efficiency can be further improved.

Further, by supplying high-concentration oxygen produced by an oxygen production apparatus to the thin tube, necessary and sufficient oxygen can be supplied to the culture solution.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing the overall configuration of the cell culture device, and FIG. 2 is an explanatory diagram showing the flow of a culture solution in a culture tank. DESCRIPTION OF SYMBOLS 1 ... Cell culture apparatus, 2 ... Supply part, 3 ... Culture part, 5
... culture tank, 6 ... stirring blade, 7 ... gas supply body, 7a ...
Thin tube, 16a …… Oxygen production equipment, G …… Gas supply system

Claims (3)

(57) [Claims] 1. A cell culture apparatus for supplying a gas such as oxygen necessary for culturing cells to a culture solution and stirring the culture solution to disperse the cells in the culture solution and performing cell tissue culture. The supply of the gas is performed by a thin tube made of a gas-permeable polymer material, and the thin tube is swirled or assembled to form a cylindrical gas supply body having both ends opened, and the gas supply body is used as a culture solution. A cell culture device, wherein the opening is arranged up and down at the center of the cell culture device. 2. The cell culture apparatus according to claim 1, wherein the stirring of the culture solution is performed by a stirring blade disposed on a center axis of the cylindrical gas supply body. 3. The cell culture device according to claim 1, wherein oxygen produced by an oxygen production device such as a pressure fluctuation type oxygen production device is supplied to the thin tube made of the polymer material.