JPH0499483A - Method for perfusion culture of animal cell - Google Patents

Abstract

PURPOSE:To culture animal cells in a high density for a long period by taking out a suspensible animal cell-suspending culture solution, separating the cells from the culture solution with a flat plate filtering membrane intermittently washed and having a specific pore diameter and subsequently returning the Separated cells together with a fresh medium to a culture tank for the continuous exchange of the medium. CONSTITUTION:In a method for perfusion-culturing animal cells by charging a medium in a stirring culture tank BR, adding the animal cells in the medium, aerating the medium with air containing 5% of CO2, simultaneously stirring the culture tank slowly for culturing the cells, taking out a part of the culture solution suspending the suspensible animal cells, separating the animal cells from the culture solution by a filtering system equipped with a flat plate filter membrane having pores of 0.2-0.8mu and subsequently returning the animal cells together with a fresh medium to the culture tank BR for the continuous exchange of the medium, the flat plate filter membrane is intermittently washed with the medium in the same direction as the circulation direction of the culture solution, thereby permitting to subject the suspensible animal cells to a continuous perfusion culture for a long period.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for culturing planktonic animal cells at high density for a long period of time.

[Prior art] Animal cells can be classified based on their growth characteristics into cells that proliferate in suspension, such as lymphoid cells, and cells that proliferate by adhering to the surface of culture vessels or collagen matrices. . Since there are many floating cells such as hybridomas that are useful for material production, many methods for mass culturing have been devised.

-The biggest challenge in cell culture for boat fishing is how high the density can be raised per 1 d of culture solution. Under normal culture conditions, in the case of animal cells, culture solution 1I21! It is about 105 to 106 pieces per serving. For this purpose, by periodically exchanging the culture solution and performing gas replacement,
It is known that it is possible to achieve cell densities of 6-10''.

For this purpose, a method for continuously exchanging the culture medium is to constantly circulate and perfuse the culture medium, exchange the culture medium in this circulation circuit, and also collect useful substances produced in the medium. being developed. Initially, this technology was used for high-density culture of microorganisms.
Publication No. 3) discloses a method for high-density culture of lactic acid bacteria. According to this method, the culture solution is extracted from a lactic acid bacteria culture tank, low-molecular substances and bacterial cells are separated using a hollow fiber filtration membrane, and the bacterial cells are returned to the culture tank together with a fresh medium. It is characterized by backwashing. As an example of applying this method to planktonic animal cells, JP-A-61-257181 describes culturing antibody-producing hybridomas, using a hollow fiber filtration membrane, exchanging the medium, and filtering through the hollow fibers. A method is disclosed in which the membrane is periodically backwashed with serum-free medium. According to this method, hybridomas were cultured for 22 days and a cell density of 1.3 x 10' cells per 1111 culture fluids was reached. In this way, in planktonic cell culture, perfusion of the culture medium, exchange of the medium with a hollow fiber membrane, and backwashing of the hollow fiber membrane can be said to be important points in high-density culture. However, although this method of using hollow fiber membranes is effective in increasing the membrane area of the device, the culture medium containing cells
It has been pointed out that because it flows through the tubules of hollow fibers, strong shearing force is applied to cells, causing cell disruption. In addition, cells often adhere to the filter surface, causing clogging, making long-term culture difficult. For this reason, in order to achieve the effect of backwashing, it has been common practice to use a filtration membrane with small pores that have the ability to cut molecular weights of 10,000 or less.

However, when membranes with pores of this size are used, problems have been pointed out, such as the inability to efficiently separate and recover polymeric substances such as proteins and antibodies secreted into the culture medium.

By the way, IgG, which is a typical subclass of antibodies, is M
Many useful substances have high molecular weights, such as W146,000 and 69,000 for t-PA.

[Problem to be solved by the invention]

The present inventors have investigated long-term continuous culture methods and material production for planktonic animal cells, and have discovered an optimal combination of a new medium exchange and filtration membrane that improves the drawbacks of hollow fiber filtration. This led to the completion of the present invention.

Therefore, an object of the present invention is to provide an optimal culture method for continuously culturing planktonic cells for a long period of time using a filtration membrane.

[Means to solve the problem]

In the present invention, when continuously culturing planktonic animal cells for a long period of time, a part of the culture solution in which the cells are suspended is removed, the culture solution and cells are separated through a filtration membrane, and the cells are placed in a culture tank together with a fresh medium. This is a culture method in which the culture medium is continuously exchanged by returning the cells, and (1) using a flat filter membrane with pores of 0.2 μm to 0.8 μm for cell separation; Specifically, it has been found that it is possible to culture target cells at high density for a long period of time by performing a washing operation in the same direction as the circulation direction of the culture solution.

In order to perform culture according to the method of the present invention, a culture apparatus having the functions shown in FIG. 1 can be used as an example.

In a culture tank indicated by BR, floating animal cells are gently stirred and cultured using a stirrer (Sll). The cell suspension is introduced into the filtration system (FM) through a line designated Ll and constantly through a pump designated Pl. And the flat plate II installed inside this
The culture filtrate and the culture concentrate containing cells are separated by filtration using the T18 membrane.

The culture concentrate containing cells is returned to the stirred culture tank (BR) through line L2. In addition, the culture solution extracted from the stirred culture tank (BR) is detected by a liquid level gauge (LS).
A decreasing amount of the medium is supplied from the supply medium (SM) through the pump (B2) and kept constant in the stirred culture tank (BR). The pH and O□ concentration in the stirred culture 1 (BR) are detected by a dissolved oxygen meter (DO) pH controller,
Always controlled to a steady value.

As a filtration system for separating cells and culture fluid, any system can be used as long as it is equipped with a flat filtration membrane. The pore size of the filtration surface is preferably one that has a membrane that does not allow floating animal cells to pass through but allows macromolecular proteins to pass through.From this point of view, the pore size is as follows:
Particularly preferred is 0.2 μm to 0.8 μm.

During perfusion, the culture solution is sucked by the pump (B3) and after filtration is collected in the recovery medium tank (RM) through the line (LS), and sent to the purification and recovery step as required.

In conventional filtration devices using hollow fibers, where the surface of the filtration membrane is cleaned periodically (intermittently) in the same direction as the circulation direction, it is difficult to clean the membrane surface, and clogging inside the hollow fibers occurs. However, in the flat membrane filtration method of the present invention, washing should be performed once a day in the same direction as the circulation direction with a basal medium containing the same components as the culture solution. It was found that continuous culture could be maintained for a long period of time.

For cleaning, stop the pumps (PI) and (B3), switch the valves (B2) and (B3) to the cleaning line, drive the pump (B4), and increase the perfusion flow rate of the cleaning liquid from the cleaning liquid tank (CM). Pulp (B2), (B3)
What is necessary is to switch the pumps (Pi) and (B3) and restart perfusion. By adopting such a method, long-term, high-density culture of planktonic animal cells and recovery of the culture medium become possible.

The flat plate filtration membrane in the present invention is a microfiltration membrane (10-'
11-30 to 50 micron filtration capacity) in the form of a single layer or multi-layer and formed into a sheet, such as Millipore filter (Millipore), Ultra filter, Ioplate (Amicon), etc. I can give an example. In addition, examples of filtration systems that include these filters include Minitan (Millipore), Cellprosensor (Amicon), Ioplate System (Amicon), etc. When using hollow fibers, It is not necessary to backwash the cleaning solution as in the above method, and cleaning can be easily performed, and cell crushing, membrane surface clogging, etc. can be prevented.

The present invention also provides FM3A, lymphocytes, Namalva cells,
It is used to culture animal cells such as hybridomas, and the physiologically active substances produced by these cells can be efficiently collected.

The method of the present invention will be explained in more detail below with reference to Examples.

Example An example in which floating cells FM3A were cultured using the culture apparatus shown in FIG. 1 is shown.

FM3A is a mouse-derived cell line established by Koyama et al. In Japan, it is registered as JCRBO701 in the Cancer Research Foundation Research Resource Bank and is easily available.

DMEM (Dulbecco modi) containing 5% calf serum in a stirred culture tank (BR) (used with a capacity of 11).
fiedEagie 5edius) 500-,
FM3A was added to this so that the initial cell density was 1X10'/d, and culture was started. Culture was carried out while gently agitating the culture tank at 40 rpm while air containing 5% CO2 was passed through the liquid surface.

The culture solution was circulated at a rate of 700-800 d/hr.
In addition, the permeation collection by the culture solution filtration system is 60 d/h.
The amount was r. The level sensor (L) detects the decrease in the culture solution.
5%C is detected automatically from line (B4).
3-containing DMEM was supplied.

Valve the line (B2) every 24 hours after starting culture.

Switch to the cleaning line by (B3) and pump (Pl),
(B3) and drive the pump (B4) to remove serum-free D.
? The filtration system was cleaned using 1EM for 450 d at a rate of 1000 d/5 in (50-100 times the circulation flow rate) in the same direction as the perfusion.

The filtration system used in this example was Minitan manufactured by Millibore (pore size of 0.65 μm). When cultured using this method, the results shown in Figure 2 (
-0-○-), the maximum cell density exceeded 3×10′, and long-term culture of 32 days or more was possible. As a comparative example, the culture results were shown when this device was used and only fresh culture medium was supplied without washing the membrane surface (-low-low).

In this case, a cell density of 10'/d was reached for a short period of time (10 days), but the cells slowly died thereafter and could only be maintained for 15 days.

In addition, the results of batch culture performed in T-flasks (-△
−△−). DMBM containing 5% C8 was used as a medium, and culture was performed in an incubator under a 5% CO8 atmosphere. The cell density reached only 2×10′/d, and the culture could only be maintained for 8 days.

Thus, it was confirmed that floating cells could be cultured at high density for a long period of time by the method of the present invention. In addition, 1,500 af of culture fluid could be collected every day.

〔Effect of the invention〕

By implementing the present invention, high-density, perfusion culture of planktonic animal cells has become possible. According to this method, culture medium can be exchanged and recovered continuously, and cells can be cultured in good conditions without clogging of filter holes or crushing of cells, which were conventional problems.

[Brief explanation of drawings]

FIG. 1 schematically shows the steps of the method of the invention. FIG. 2 shows the results of long-term culture of floating cells FM3A according to the method of the present invention. Two examples of culture results obtained using conventional methods are also shown for reference. In the figure, −〇−〇~ indicates the culture results according to the method of the present invention, -　~low indicates the culture results according to the conventional method (circular culture method), and -△
-△- indicates the culture results by the conventional method (batch culture method). Cell density [cells/ml]

Claims (2)

[Claims] (1) Remove a portion of the culture solution in which planktonic animal cells are floating, separate the culture solution and animal cells using a filtration membrane, return the animal cells to the culture tank along with the fresh medium, and continuously exchange the medium. In the long-term continuous culture method, [1] a plate filtration membrane having pores of 0.2 μm to 0.8 μm is used for cell separation, and [2] the plate filtration membrane is intermittently passed through the culture medium in the direction of circulation of the culture solution. A method for long-term continuous perfusion culture of planktonic animal cells, which is characterized by washing from the same direction. (2) Claim (1) wherein the planktonic animal cells are FM3A cells.
) Long-term continuous perfusion culture method of planktonic animal cells.