JPS5959187A - Method and apparatus for cell culture - Google Patents

Abstract

PURPOSE:To culture adherent cells in high efficiency, by proliferating the cells on the substrate without immersing the substrate in culture liquid while contacting the substrate with the culture liquid. CONSTITUTION:The substrate container 3 is fully packed with the substrates 1, the culture liquid is sprayed from the sprayer 4 placed at the top of the culture tank 2 to effect the thorough contact of the liquid with the substrates 1, and the culture liquid dripped to the bottom of the container 3 is collected in the tank 20 and circulated to the sprayer 4 with a pump. The substrate container 3 is aerated with a gas having necessary composition simultaneously to the initiation of the culture. The gas is introduced into the culture tank 2 from the gas diffuser 17 attached at the top or bottom of the culture tank and made to contact countercurrently or concurrently with the culture liquid. The adherent cells can be proliferated on the substrate which is not immersed in but contacted with the culture liquid, in high efficiency.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method and apparatus for culturing adhesive cells.

More specifically, the present invention relates to a method and apparatus for efficiently culturing adherent cells over a wide area of the surface of a support.

In general, cell culture is used for the production of santonins derived from plant cells, and for the production of interferon, urokinase, and various hormones derived from animal cells.

Furthermore, cell culture is an effective technique for producing various biological substances or for basic research on living organisms.

However, in cell culture, it is essential to grow the cells that produce the target substance in large quantities.
For this reason, much research has been done into searching for growth factors for cells and into techniques for mass culturing cells.

Conventional cell culture methods can be broadly classified into suspension culture methods and monolayer culture methods. In the suspension culture method, agitation is performed to prevent cells from settling, but agitation does not damage the cells, and since it is a deep culture, stirring is essential for cell growth. Bubbling occurs when oxygen is supplied, making culturing difficult, and requires complicated operations, such as taking measures to protect the culture from contamination.

On the other hand, currently known monolayer culture methods include the roller culture method and the multilayer plate method, but all of these methods have limitations on the area in which cells can grow, and they also prevent gas from coming into contact with the cells. There were some difficulties.

As a result of research for an effective and simple cell culture method, the present inventors were able to solve the problem by employing a support for cell culture.

The present invention provides a cell culture method and device, characterized in that cells having adhesive properties are grown on a support that is immersed in a culture solution, and the culture solution is adhered to the support and cultured. It is related to.

The present invention overcomes the disadvantages of the microcarrier method, such as damage to cells caused by agitation and foaming caused by aeration, and allows for continuous culture, with no need to change or replenish the culture solution, or adjust the amount of aeration or gas components. This is a method and device for mass culturing cells that can be extremely easily controlled.

A major feature of the present invention is that a support is used in cell culture. The support may be made of polystyrene, steel, glass, aluminum, rubber, polypropylene, polycarbonate, polyethylene, Teflon, or any other material to which cells can adhere. To facilitate this, K may also carry out coating of the support with an effective substance.

The support may be of any shape, such as a rod, sphere, plate, fiber, or various deformed shapes, but one of the shapes that are effective in the present invention is the tassel-bridge shape shown in FIG. There is a support. This tassel-shaped support has an extremely large surface area per unit volume, and when it is used after being broken or filled, there is no place where the culture solution gets mixed up, making it extremely effective.

This support is housed in a support storage container within the culture tank.

The culture solution is sprayed from the culture solution dispersion device installed at the top of the culture tank so as to be in direct contact with the support, and the culture solution that falls downward collects in the tank and is sent to the spraying device by a pump.
It is designed to be circulated.

At the start of culture, set the culture tank to a temperature suitable for cell growth.
While keeping the temperature at 0 to 40°C, it is filled with a culture medium in which predetermined cells are dispersed, and after a certain period of time, circulation is started and the cells are attached to the support surface to start proliferation.

Simultaneously with the start of culture, a gas having a required gas composition is aerated, if necessary. The lower part of the culture tank is also vented through a gas dispersion device provided at the upper part, and the gas composition or the amount of aeration is set to be effective for the target cells. The gas introduced into the culture tank flows through the pores of the support and moves countercurrently or cocurrently with the culture solution. At this time, the circulating culture solution falls on the cell surface on the support in a thin layer, but this culture solution and gas come into contact with each other efficiently, and the gas contact area is the same as the cell adhesion area. Because of this, the gas components are transferred into the culture solution extremely efficiently.

As the culture progresses, cells proliferate over the entire surface of the support, and at the same time various products begin to be eluted into the culture medium.

Here, a portion of the culture solution is constantly withdrawn, and the same amount of fresh culture solution is added, and the culture is continued.

If the amount of exchange between old culture solution and fresh culture solution is set to an appropriate amount, continuous operation can be performed for a fairly long period of time while the cells in the culture tank remain active.

Serum is often used in cell culture media, but adding serum makes it extremely difficult to recover small amounts of biological substances produced by cells.

However, in the present invention, since the culture solution can be exchanged very easily, it is possible to switch to the culture solution for substance production midway through the process, making it easy to separate the product. Regarding ventilation, it is necessary to change the gas composition depending on the properties of the cells, but in the present invention,
The gas composition can be freely set, and since it passes through the support voids, there is no need to worry about foaming as in the conventional case, and there is no need to worry about bubble dispersion or surface area, which is a problem when gas is aerated into the culture solution. There is no need to consider the increase in

Generally, cells have a limited period of activity, so it is necessary to inactivate the cells and to proliferate cells with other activities. In the present invention, when the production amount of the target product decreases, a solution having a cell detachment effect, such as a trypsin solution, is sprayed in place of the culture solution, and then a fresh culture solution is circulated and the cells remain on the support. By allowing some of the cells to proliferate, the culture tank can be used continuously by newly proliferating cells.

1. If you want to produce a large amount of a target substance, use a multi-vessel system and sequentially activate the cells, making it possible to culture efficiently and stabilize the overall productivity.

To explain the device of the invention with reference to the drawings, FIG. 1 shows a support 1. In FIG. FIG. 2 is a schematic diagram of the culture device, and FIG. 6 is a longitudinal cross-sectional view of the culture device.

? 3 indicates a culture tank, and 3 is a support housing chamber, which is completely filled with the support 1. A large number of pores 5, 5, etc. are opened using a 4-cell culture solution dispersing device, and the culture solution sent therein is sprinkled onto the support in a shower-like manner. Reference numeral 6 denotes a perforated plate, on which a gas blowing lower is appropriately provided. Reference numeral 8 represents a culture tank wall, and reference numeral 9 represents a wire mesh, which together form a support housing chamber 6. 10 is a jacket,
Hot water is introduced from a hot water outlet 11 and discharged from a hot water outlet 12 to heat the culture tank. , 15 is a perforated plate, in which a gas outlet 14 is appropriately provided. A gas blowing main pipe 16 is provided in the center of the culture tank bottom 15,
The gas introduced through the gas introduction pipe 17 is blown out. 18 is a culture solution introduction pipe, 19 is a culture solution discharge pipe, and the culture solution put in the tank 20 is sent from the culture solution introduction pipe 18 to the culture tank, passes through the culture solution discharge pipe 19, and is transferred to the tank 2.
Cycles to 0. The tank 20 is heated to about 20 to 40°C by hot water sent from a water heater 21. A part of the circulating culture solution is drawn out and put into the sampling tank 22, and a fresh culture solution is put into the culture solution tank 23, so that the part that has been drawn out is replenished.

In addition, carbon dioxide gas flows from the carbon dioxide gas cylinder 24 to the flowmeter 25.
It is taken in from the pump 26, passed through the flow meter 27, mixed with air washed with acid 28 and alkali 29, heated to about 67°C by the warming machine 31, and then gently inserted into the gas introduction pipe 17 to culture. introduced into the tank. The discharged gas is circulated through the discharge pipe 32, and a portion passes through the filter 33 and is discharged into the outside air.

Next, examples of the present invention will be shown.

Example 1 Eagles' minimu in a plastic petri dish
L cells cultured in messential medium (MEM) with 1% bovine serum added to 0.125
Detach the cells with % trypsin solution (containing BDTA),
Wash the cells with the same culture solution.

After suspending the washed L cells in the same culture solution, they are aseptically injected into a large plastic petri dish containing gas-sterilized filling material and cultured for 24 hours in an incubator containing 5% CO2.

Thereafter, the same culture solution was circulated in the culture tanks shown in FIGS. 2 and 3 filled with the support shown in FIG. 1, and K 5 % C.
Vent with O2. At this time, the culture solution is kept warm at 37°C and warm water at 37°C is circulated through the jacket.

Approximately one week after circulating the same culture solution under 5% CO2 aeration, the surface of the filling material is covered with cells. The amount of circulating culture solution is Q, 1 V/V/M, and the amount of aeration is 0.05 V/
V 7M To LJ,=.

Example 2 Dulbeccos' Mod in a plastic petri dish
ifiedEagle Medium (DMB)
3T3 cells cultured in a culture solution containing 10 g of bovine serum (0.125%) were detached with an IJ pushin solution (containing EDTA), and the cells were washed with the same culture solution. washed 6
After suspending T3 cells in the same culture solution, they were aseptically injected into a large plastic petri dish containing gas-sterilized filling material.
Culture in an incubator containing %C02 for 24 hours.

Thereafter, the same culture solution was circulated in the culture tank shown in FIGS. 2 and 3 filled with the support shown in FIG. 1, and 5% Co was aerated therein. At this time, the culture solution was kept warm at 37°C and kept warm by circulating warm water at 37°C in a jacket.

Approximately one week after circulating the same culture solution under 5% CO2 aeration, the surface of the filling material is covered with cells. The amount of circulating culture solution was 0.1'J/V/M, and the amount of aeration was 0.05.
V/V/M.

[Brief explanation of drawings]

FIG. 1 is a diagram showing an example of a support, FIG. 2 is a schematic diagram of the device of the present invention, and FIG. 3 is a partially sectional view of the device of the present invention. 1... Support? ... Culture tank, 5... Pore, 6.
...eye plate? ... wire mesh agent patent attorney door 1) parent male number 11! l

Claims (3)

[Claims] (1) A cell culture method, which comprises growing adhesive cells on a support that is not immersed in a culture solution, and culturing the support by bringing the culture solution into contact with the support. (2) The cell culture method according to claim 1, wherein a gas having a necessary gas composition is brought into contact with the culture solution by aeration on the support in a countercurrent or cocurrent manner. (3) A cell culture device consisting of a culture tank having a support housing chamber through which culture solution and gas can pass, a culture solution spreading device installed at the top of the culture tank, and a gas dispersion device installed at the bottom or top of the culture tank. .