JPS6147184A - Cumulative suspension culture for somatic cell and culture medium useful for same - Google Patents

Abstract

PURPOSE:To make cumulative cells into a slow suspension state, and to cultivate them efficiently without damaging them, by dividing a culture meidum into a cell retaining tank and a medium tank by a porous member to pass a medium but not to pass the somatic cells, feeding continuously the medium and stirring the medium. CONSTITUTION:Somatic cells are collected and kept in one of the tank (somatic cell-retaining tank) 2 of a culture medium divided by the porous member (e.g., Teflon perforated plate ) 3, and a mediun in the other tank 1 is sitirred, so that cumulative cells in the cell-retaining tank 2 is made into a slow suspension state, a fresh medium is fed to the culture tank 1, the medium is transferred to the cell-retaining tank 2, and the consumed medium is discharged from the medium outlet 9. Part of the medium and a fresh medium are fed to the medium tank 1 and circulated. By using the culture medium of the construction, the cumulative cells are made into a slow suspension state, and high-density cell culture is carried out.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a somatic cell enrichment suspension culture method and a culture vessel used therein.

[Prior Art] The principle of the Odori culture method for somatic cells is to expand the culture volume for floating cells and the culture surface area for adhesion-dependent cells. To date, the method has been implemented in low-speed agitated culture vessels for floating cells and attachment-dependent cells attached to microcarriers (e.g., spinner culture flasks, low-speed jar fermenters, etc.).
.

This low-speed stirring incubator allows the culture solution in the incubator to be stirred at low speed (5~
By stirring at a speed of about 30 times/min), floating cells and cells attached to microcarriers are suspended and suspended, increasing the culture volume, and at the same time, increasing the number of contacts between the culture medium and cells, thereby reducing the absorption rate of culture components. This is something we are trying to improve.

[Problems to be Solved by the Invention] However, for both floating cells (non-adhesion-dependent cells) and attachment-dependent cells, the agitation operation in the conventional culture method described above creates a shear force between the culture medium and the cells. This causes damage to cells and inhibits their proliferation to some extent.

That is, in the case of non-adhesion-dependent cells, direct damage occurs due to the shear force generated by agitation, and in the case of attachment-dependent cells, the shear force causes microcarrier-11JI! Dissociation of the complex and direct damage to adherent cells occurs.

[Means for solving the problem 3] The present inventor has completed the present invention by intensively researching a suspension culture method that can reduce damage to cultured cells and a culture vessel used therein.

That is, in the first method of the present invention, somatic cells are accumulated and retained in one tank of a culture vessel partitioned with a porous member, and the culture medium in the other tank is stirred. This is a somatic cell enrichment suspension culture method characterized by culturing accumulated cells in a slow suspension state, and the invention of the second method is the result of studying high acid density culture, and is different from the first method. In the invention, a fresh medium is supplied to a culture medium tank, the medium is transferred to a cell holding tank, and the consumed medium is discharged from the cell holding tank for continuous cultivation. This is a somatic cell enrichment suspension culture method that is characterized by: The invention of a third method is the invention of the second method, characterized in that a part of the medium discharged from the cell holding tank is supplied to the medium tank together with a fresh medium, and the culture is carried out while circulating the medium. This involves a cell enrichment suspension culture method.

Further, the invention of the apparatus of the present invention for carrying out the invention of the method described above is such that the inside of the culture vessel is partitioned into a cell holding tank and a medium tank by a porous member that allows the medium to pass through but does not allow the cells to pass through. This invention relates to a suspension culture device for accumulating somatic cells, characterized in that the culture medium in the culture medium tank can be stirred.

In the above, the porous member that partitions the culture vessel may be any member having a large number of pores that allow the culture medium to pass through but not the cultured cells. Specifically, mesh cloth such as nylon mesh can be used.

The size and shape of the culture vessel to be used can be appropriate depending on the purpose of culture, quantity, etc. The culture vessel can be partitioned vertically, horizontally, or diagonally, but it is best to partition it vertically. is preferable.

Any tank in a partitioned culture vessel may be used as a cell holding tank, but when partitioned into an upper and lower tank, it is preferable to use the upper tank as the cell holding tank and the lower tank as the culture medium tank.

A spinner or the like can be used as a means for stirring the medium in the culture medium tank.

Body III cells that can be applied to the culture method of the present invention include non-adhesion-dependent floating cells and attachment-dependent cells. Specifically, plasmacytoma (SP) is an example of floating cells.
210), and hybridomas created by a cell fusion method with tile cells of BALD/C mice immunized with SP 210.111 cells and distemper virus.

Examples of adhesion-dependent cells include Vero cells, distemper virus, etc., in which case microcarriers are used as adhesion carriers.

The medium to be used is appropriately selected depending on the type of somatic cells to be cultured, and other culture conditions (culture temperature, etc.) are carried out under normal general conditions.

[Effect] The first method is the so-called batch culture method,
Since the cell holding tank and the medium m<stirring WJ) are isolated by the II saturation holding membrane made of a porous member, the medium is forcibly flowed by the stirring force in the stirring tank, and the cell holding tank is passed through the porous member. Accumulated cells such as floating cells in the tank and microcarrier/adhesion-dependent cell complexes are suspended in a slow floating state. That is, since it is not necessary to stir the entire culture medium in the conventional culture tank, it is possible to reduce the adverse effect of 1tA damage on the cells due to this stirring.

The second method is the continuous i8 cultivation method, in which fresh medium is supplied from a stirring tank, the fresh medium is passed through the cell culture tank, and the spent medium is discharged from the cell holding tank. ) It is possible to obtain about 8 to 10 times more cells than the method.

The third method is a circulating continuous culture method in which the medium is continuously circulated in the continuous culture method, which can dramatically improve virus culture.

Furthermore, according to the incubator of the present invention, each of the above-mentioned culture methods can be carried out extremely easily.

[Example] EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 FIG. 1 shows an example of the incubator of the present invention, in which a cylindrical cell holding tank 2 is positioned above a cylindrical culture medium tank 1, and a deflon eye is placed between both tanks 1 and 2. A filter 4 sandwiched from both sides by a plate 3 is interposed, and both tanks 1 and 2 are fixed by a fixing ring 7 through an O-ring piton 5 and an O-ring 6 so as to be watertight. 8 is a medium inlet provided on the side of the medium tank 1, 9 is a medium outlet provided on the upper side of the cell holding layer 2, 10 is a cap, 11 is a packing, 12 is a magnetic stirrer 13 at the bottom of the medium tank 1. The screw type magnet rotated from the outside is not shown.

Non-adhesion dependent cells such as plasmacytoma (SP21
0), a filter 14 of 60 to 90 mm is installed, and the stirring speed of the medium WJ1 is set to 35 to 50 mm.
By setting the rate to 0 times/min, the cells on the filter 4 can be placed in a slow floating state. The scale shown in the M1 diagram (total height 3
According to the incubator of the present invention having a diameter of 50 mm and a diameter of 110 mm, the cell connection rate is
11e x 1 (111tLjC'1 combination, 10 6-3 cells/1! after 10 days of culture, approximately 10 times the proliferation rate compared to 105.5 cells/11 in conventional spinner culture) I can do it.

Example 2 Even more effective is the case where products of somatic cells are obtained.

For example, by culturing BAL8/c mouse tile cells immunized with 5P2101 cells and distemper virus and hybridomas produced by the cell fusion method in the incubator of the present invention shown in FIG. Monoclonal antibody productivity can be improved compared to using the same volume of culture medium in a spinner flask.

That is, 1! During 7 days of culture in spinner flasks (rotation speed: 3 orpm, D-MEM as medium, 1.0% fetal bovine serum), anti-distemper monoclonal antibodies
Whereas only 0 unit/ν1 (S/N value) is produced, the culture method of the present invention produces 5200 units/W (S/N value).
1q, indicating that the present invention improves the physiological state of the cells compared to conventional spinner culture.

Embodiment 3 FIG. 2 shows another embodiment of the incubator of the present invention, which has substantially the same configuration as the previous embodiment, in which a pipe 16 from a fresh culture medium container 15 is connected to a culture medium 8, and a tube 16 from a fresh culture medium container 15 is passed through a bacterial filter 17. Pressure gas is fed into the fresh culture medium container 15 to supply fresh culture medium to the culture medium tank 1.

In order to perform efficient culture using 11 heavy ring vessels, the second
As shown in the figure, a continuous culture method can be used by injecting a fresh medium from the medium tank 1 and discharging it from the cell holding tank 2 in which the cells are being cultured. Compared to the batch method, the continuous culture method allows for the dilution of IIIVI and growth inhibitory substances as well as the supply of fresh nutrients, for example 3.5! By continuously culturing the hybridoma cells, 36,480 units # (S/N value) can be obtained in a total of 7 days.

Example 4 The incubator of the present invention also exhibits the following effects when culturing adhesion-dependent 1 cells adhered to a carrier. Cytodex (CVt0deX) in the incubator shown in Figure 1 21. :
When culturing the attached a (Vero) m cells (Ill cells/carrier complex), the proliferating Vero cells on the microcarriers are grown in a normal spinner flask (
1)), the culture medium of the present invention
81IIIli in law! / It can be improved into a carrier.

Example 5 When distemper virus is cultured in the same culture system as in Example 4, spinner culture yields 107/yf, but the culture method of the present invention yields 1o910yf.

Embodiment 6 FIG. 3 shows still another embodiment of the incubator of the present invention, in which the conduit 18 is connected to the medium outlet 9 of the cell holding tank 2, and the cough tube The tip of the channel 18 is branched, one branch pipe 19 is used for discharge, and the other branch pipe 2 is used for discharge.
0 is used for circulation, and a circulation pump 21 is used to circulate and supply the culture medium together with the fresh culture medium to the culture medium tank 1.

With this incubator, a circulating continuous culture method can be performed and distemper virus can be produced at a high level.

For example, when Vero cells are grown with Cytodex 2 at a concentration of 25 ma/yf, 3.5! A total amount of virus of 1 t3" x 3.s! can be obtained by carrying out continuous circulating culture.

[Effects of the Invention] As described above, according to the somatic cell enrichment suspension culture method of the present invention and the culture vessel used therein, the inside of the culture vessel is partitioned with a porous member, and one tank is a 1oIll holding tank and the other tank is By using the medium tank as a culture medium tank, by stirring the culture medium tank, the gentle stirring effect spreads to the cell holding tank for culture. Damage to Ill cells can be significantly reduced and efficient culture can be performed. In addition, by continuously culturing while supplementing fresh medium, high-density cell culture can be achieved, and by culturing while circulating a portion of the medium, cell products in the culture solution can be The concentration can be significantly increased.

[Brief explanation of the drawing]

FIG. 1 is a partially cutaway explanatory diagram of an embodiment of the incubator of the present invention;
FIG. 2 is an explanatory diagram of another embodiment of the incubator of the present invention, and FIG. 3 is an explanatory diagram of still another embodiment of the culture medium III of the present invention. 1 is a medium tank, 2 is an m cell holding tank, 4 is a filter, 8 is a medium inlet, 9 is a medium outlet, 12 is a screw type magnet, and 15° is a fresh medium container. Patent application Nippon Zenyaku Kogyo Co., Ltd. Figure 1 Figure 2

Claims (1)

[Claims] 1) Somatic cells are accumulated and maintained in one tank of a culture vessel partitioned with a porous member, and the accumulated cells in said one tank are slowly removed by stirring the medium in the other tank. A somatic cell enrichment suspension culture method characterized by culturing in a suspended state. 2) The somatic cell enrichment suspension culture method according to claim 1), wherein the cells accumulated and maintained in one tank are floating cells. 3) The somatic cell enrichment suspension culture method according to claim 1), wherein the cells accumulated and retained in one tank are adhesion-dependent cells adhered to a carrier. 4) Somatic cells are accumulated and maintained in one tank of a culture vessel partitioned by a porous member, and the culture medium in the other tank is stirred to bring the accumulated cells in the one tank into a slow floating state, and the other tank is A somatic cell enrichment suspension culture method characterized by supplying a fresh medium to the tank, transferring the fresh medium to the one tank, discharging the consumed medium from the one tank, and culturing continuously. 5) Somatic cells are accumulated and maintained in one tank of a culture vessel partitioned by a porous member, and the culture medium in the other tank is stirred to bring the accumulated cells in the one tank into a slow floating state, supplying a fresh medium to the tank, transferring the fresh medium to the one tank, discharging the consumed medium from the one tank, and supplying a part of the discharged medium to the other tank together with the fresh medium. A somatic cell enrichment suspension culture method characterized by culturing while circulating a medium. 6) Somatic cells, characterized in that the inside of the culture vessel is partitioned into a cell holding tank and a medium tank by a porous member that allows the medium to pass through but not the cells, and the culture medium in the medium tank can be stirred. Integrated floating culture vessel. 7) The cell holding tank is a cell holding tank equipped with a medium outlet,
The somatic cell enrichment suspension culture device according to claim 6, wherein the culture medium tank is a culture medium tank equipped with a medium inlet.