JPH0240318B2 - - Google Patents

Abstract

PURPOSE:To enable the (mass) culture of cells having higher specific gravity than the culture medium, e.g. animal cells sensitive to shearing force, etc., by precipitating at least a part of cells in a culture liquid in a culture tank of a bubble-tower perfusion culture apparatus and adding fresh culture medium to the tank while discharging the supernatant liquid of the culture liquid. CONSTITUTION:(A) A serum-free medium is produced e.g. by adding transferrin, insulin, sodium pyruvate, selenous acid, galactose, glutamine, HEPES, penicillin, streptomycin and sodium bicarbonate to RPMI-1640 medium and (B) namarva cell which is a human lymphoblast cell is cultured in the medium A. The medium A is charged into a culture tank 13 of a bubble-tower perfusion culture apparatus through a medium inlet port 3, the cell B is inoculated in the medium via an inoculation port 2, the medium is aerated with air and air containing 5% CO2 at a specific flow rate through an aeration line 9 and the cell B having higher specific gravity than the medium A in the culture liquid is precipitated. The produced supernatant liquid is separated in a supernatnat liquid-separation tank and discharged through an effluent line 11. At the same time, the medium A is successively added to the medium through a medium inlet port 3 to continue the culture of the cell.

Description

[Detailed Description of the Invention] Industrial Application Field The present invention is applicable to animal and plant cells, tissues, organs, etc.
The present invention also relates to a bubble column perfusion culture method suitable for culturing microorganisms, particularly cells having a higher specific gravity than the medium used, and an apparatus therefor.

BACKGROUND ART Conventionally, most of the apparatuses used for culturing animal and plant cells and organs and culturing microorganisms are of batch type. When such a batch-type device is used, the culture is terminated when the nutrient source in the medium introduced into the culture tank is used for cell proliferation, etc. and is depleted. Therefore, in order to obtain a large number of cells, a large-sized device is required.

In the case of batch culture, the most common method is aeration and agitation culture, in which stirring and aeration are carried out using an impeller and spargeer, and the bubble method, in which rotational movement is caused by air bubbles for agitation.There are culture devices suitable for each. Are known. Among these, the bubble column culture device is a culture device that performs aeration and stirring by aerating air etc. from the bottom of the culture device [Iden 38 (8), 72-79 (1984)] .

However, in the batch culture method, the pH within the culture system
It is difficult to achieve a viable cell density above a certain level due to factors such as a decrease in cell density, inhibition of growth by metabolites, and depletion of nutrient sources. Recently, perfusion culture devices have been developed that can provide higher cell densities than batch culture. A perfusion culture device is a culture device that continuously supplies a culture medium and withdraws a culture supernatant. In this type of culture device, cells and supernatant are separated by a rotating filter, the supernatant is discharged, and fresh culture medium is added. A rotating filter tower type or an impeller type batch culture tank is equipped with a cell sedimentation tube. Therefore, there is a cell sedimentation tube type that separates cells and supernatant, and a holofiber type that allows air to pass through a hollow fiber (hollow fiber) or a medium that passes through it [Gene 38 (8), 72-79 (1984)]. .

In addition, Japanese Patent Publication No. 36915/1986 describes a high concentration culture method for floating cells in which at least some of the cells in the culture solution are sedimented and the culture supernatant obtained is drained out of the culture device while adding fresh medium and culturing. The above-mentioned cell sedimentation tube type culture device is described as an example of a device for this purpose.

Problems to be Solved by the Invention Among the above-mentioned perfusion culture apparatuses, the rotating filter tower type and the cell sedimentation tube type are types in which stirring is performed using an impeller. However, for cells sensitive to shear forces such as animal cells, stirring using an impeller is not preferable, and stirring using a bubble column method is considered to be superior. Furthermore, it is considered difficult to increase the size of the holofiber type. On the other hand, the bubble column method can easily be made larger.

The bubble column type perfusion culture method and apparatus of the present invention can avoid such problems in conventional perfusion culture apparatuses.

Means for Solving the Problems The present invention involves adding a fresh medium while draining the culture supernatant obtained by sedimenting at least a portion of the cells in the culture medium during cell suspension culture using a bubble column to the outside of the culture apparatus. In a method for perfusion culture of floating cells and a bubble column culture device, which are characterized by culturing,
It is separated by a partition from the culture space in which the culture solution is stirred and circulated by air bubbles (hereinafter referred to as the culture tank), the upper part of the partition does not communicate with the culture tank, and the lower part of the partition does not communicate with the culture tank (hereinafter referred to as the culture tank). The present invention relates to a bubble column type perfusion culture device that is provided with a space called a supernatant separation tank, and that enables supply of a culture medium to the culture tank and extraction of the culture supernatant from the supernatant separation tank.

There are various types of bubble column culture equipment, but all types have a part where the gas flows upwards as the gas rises, and a descending part separated by a partition wall where the gas flows downwards from the top. It consists of two parts. The upward flow occurs as bubbles are generated, reaches the upper end of the partition wall, overflows, and becomes a downward flow, causing rotational movement within the tank and stirring. Cells are dispersed by this convection and uniformly supplied with dissolved oxygen and nutrients. If you create another room between these two tanks with the upper end closed and the lower end open, that is, a supernatant separation tank, the inside of this tank will be almost unaffected by agitation and will be in the same state as if it were left still. . At the upper end of the tank, where the influence of stirring is weak, cell particles with heavy specific gravity settle to the bottom and return to the stirring part of the bubble tank, resulting in a clear liquid.
Therefore, even if this clear supernatant is discharged out of the system at a slow rate, its clarity will hardly change. If the rate of discharge to the outside of the system and the rate of addition to the culture tank are the same, a constant amount of continuous culture will proceed.

In order to ensure that cell particles with heavy specific gravity that settle at the bottom end of the supernatant separation tank can efficiently return to the culture tank, the bottom end of the partition between the culture tank and the supernatant separation tank is connected to the upward and downward flow of the bottom end of the culture tank. It is preferable to provide it above the branch point.

The shape of a bubble culture tank to which such a supernatant separation tank can be added may be any shape as long as it satisfies its function, and as shown in Figures 1 and 2-1.
The types shown in and 2-2 are exemplified as preferred.

In Figure 1, the shape of the bubble column is a double cylindrical tube, and gas is emitted from the lower part of the inner cylinder at the center, causing an upward flow. The outer part is a part where the culture solution overflowing from the upper end of the inner cylinder flows downward. Furthermore, a jacket-like supernatant separation tank was installed on the outside.

Unlike FIG. 1, FIG. 2-2 shows a case in which the supernatant separation tank is provided at the center. In FIG. 3, the direction of flow of the culture solution is indicated by arrows. As shown in Figure 3-3, when the bottom of the culture tank is raised high at the center and gas is generated from below the second tank, it flows as shown by the thick arrow. Some cells are also pushed toward the center, but return to the bottom of the lower protrusion and are once again drawn into the upward flow.

In Fig. 2-1, it does not have to be cylindrical, it may be box-shaped, and three chambers can be created using separating plates. Third
-2 As shown in Figure 2, when gas is generated from the hole in the center of the lower end, it flows as shown by the thick arrow. The supernatant is discharged to the outside as indicated by the thin arrow.

As described above, a supernatant containing no cells can be separated by attaching a supernatant separation tank separated by a partition wall and having an open bottom to a normal culture tank.

Taking the apparatus shown in Figure 1 as an example, the culture volume is 2
In the case of the above apparatus, the capacity of the supernatant separation tank is approximately 1. The inner diameter of the supernatant separation tank was 110 mm, the inner diameter of the culture tank 13 was 80 mm, and the inner diameter of the inner cylinder 5 was 57 mm. The height of the apparatus is 560 mm for the culture tank 13, 370 mm for the inner cylinder 5, and 500 mm for the supernatant separation tank 10.

As shown in FIGS. 1 and 2, these culture devices are equipped with a condenser 1, an inoculation port 2, a medium addition port 3, a sampling line 4, an inner cylinder 5 or a separation plate 16, a connecting fitting 6, a packing 7, and a vent. In addition to the port 8, ventilation line 9, drain line 11, fixed parts 12, etc., there are also temperature sensors for temperature control, heaters, sensors for PH measurement and dissolved oxygen measurement, flowmeters for gas flow control, etc. Can be installed.

Generally, in order to obtain a clear supernatant liquid, it is preferable that the capacity of the supernatant separation tank is set to 0.5 to 2 for each culture tank capacity of 2.

Examples Example 1 Using the bubble column type perfusion culture apparatus shown in Fig. 1,
Culture was performed using Namalva cells, which are human lymphoblastoid cells.

Namalva cells were added at 3 μg/ml in RPMI-1640 medium.
of transfumarin, 5 μg/ml insulin,
5mM sodium pyruvate, 1.25×10 ^-8 M selenite, 1mg/ml galactose, 4mM glutamine,
10mM Hepes, 25u/ml Penicillin G, 25μg/
The cells were cultured in a serum-free medium supplemented with ml streptomycin and 0.1 g/dl sodium bicarbonate.

2 Prepare the above medium 1.9 in a culture tank, and add 1.3×
10 ⁶ cells/ml of Namalva cells were inoculated, and air and air containing 5% CO ₂ were added while adjusting the flow rate ratio appropriately.
Aeration was performed at a flow rate of 1.2-2.4/hr. 1st day 500
The medium was added from the medium addition port at a flow rate of 1/day from the 2nd day to the 8th day, and 2/day after the 8th day, and the supernatant was discharged from the drain port at the upper end of the supernatant separation tank.

Cell density was 4.1×10 ⁶ cells/ml on the 5th day, 7×10 ⁶ cells/ml on the 8th day, 1×10 ⁷ cells/ml on the 14th day,
After that, the number of cells gradually increased to 1.2× after 18 days.
It reached 10 ⁷ cells/ml.

Example 2 Namalva cells were cultured using the apparatus shown in FIG. 0.1 to the medium shown in Example 1 as a medium.
A medium was used in which pluronic F68 was added so that the concentration was adjusted to 1 g/dl. The above medium 2 was placed in a culture tank 2, and Namalva cells were inoculated at 1.3×10 ⁶ cells/ml. Aeration was performed using air and air containing 5% CO ₂ at a flow rate of 1.2 to 2.4/hr while adjusting the flow rate ratio as appropriate.

The amount of medium to be perfused is 1000ml/day after 24 hours.
Aerate until the 3rd day, then 200 liters from the 3rd day until the 10th day.
The amount was increased in ml increments to 2/day by 10 days later.
After that, increase the flow rate by 200ml every 2 days.
The day was set to 4/day. Cell count is 8x on day 8
10 ⁶ cells/ml, 1.1×10 ⁷ cells/ml on the 15th day, and 1.5×10 ⁷ cells/ml on the 20th day.

Effects of the Invention The method and device of the present invention enable the culture and mass culture of cells sensitive to shear forces.

[Brief explanation of drawings]

FIGS. 1 and 2 show a preferred embodiment of the bubble column type perfusion culture apparatus of the present invention. FIG. 3 shows the movement of the culture solution and culture supernatant in this device.

Claims (1)

[Scope of Claims] 1. In cell suspension culture using a bubble column, at least a portion of the cells in the culture solution are sedimented, and the culture supernatant obtained is discharged from the culture device while a fresh medium is added and culture is carried out. Characteristic perfusion culture method for floating cells. 2 In a bubble column culture device, a culture space in which the culture solution is stirred and circulated by air bubbles (hereinafter referred to as a culture tank) is separated by a partition wall, the upper part of the partition wall does not communicate with the culture tank, and the lower part of the partition wall is connected to the culture tank. A bubble column type perfusion culture device that is provided with a space (hereinafter referred to as a supernatant separation tank) that communicates with the culture tank, and that enables supply of a culture medium to the culture tank and extraction of the culture supernatant from the supernatant separation tank. 3. The bubble column type perfusion culture apparatus according to claim 1, wherein the lower end of the partition wall is provided above the branch point of the upward flow and downward flow at the lower end of the culture tank.