JPS60207581A - Method for cell culture - Google Patents

Abstract

PURPOSE:To enable the cultivation of a large amount of cells at a high density, by carrying out the supply of fresh culture liquid and the discharge of used culture liquid continuously or semicontinuously in a suspension-type cell culture. CONSTITUTION:The arrow shows the pipe connected to the liquid inlet and outlet connected to either of the change-over valves 12, 13, 14 and 15 in the culture vessel. The unit contains a capillary bundle composed of a number of hollow fibers. Both ends of the bundle are fixed by bonding layers, and a common inlet and outlet of the liquid is attached at one end. The other end is sealed. The unit is operated by repeating the supply of an aqueous liquid medium (e.g. fresh culture medium) and the discharge of used culture liquid alternately. Even if the wall membrane of the hollow fiber is blocked temporarily with a cell or other solid component, there occurs no permanent blocking of the tube, and the exchange of the culture liquid can be performed effectively.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a culture method for culturing and propagating cells. In more detail, it is a method suitable for carrying out cell culture on a large scale. The present invention relates to a method for culturing AJB cells in a suspension type cell culture system.

(b) Prior Art 1 Large-scale cell culture is essential for the production of viruses, vaccines, antiviral agents such as interferon, or biological drugs such as hormones. Particularly in recent years, the production of monoclonal antibodies targeting specific proteins has been based on mass culture of antibody-producing cells and myeloma/ipridoma, and solving this technology is an industrially important theme.

Traditionally, cell culture is generally done in Petri dish test tubes.

It is carried out in a laboratory model using culture bottles and the like.

On the other hand, in recent years, as a method for mass culturing cells and a device for that purpose,
Some suggestions have been made.

These proposals are based on the following methods:
ag@dependent eultur6) and suspension culture, Tsuma-shi suspension culture (5usp/n5lo
There are two types of methods: culture), and these methods are determined depending on the characteristics of the cells to be cultured.

The present invention relates to an improved method IC in suspension-type cell culture. There have been several recent proposals for culturing cells by suspension culture, for example by providing a controlled agitation function in a spinner flask by means of a magnetic stirrer or an impeller on a mechanically driven shaft. Various culture methods have been proposed.
(For example, see Japanese Patent Laid-Open No. 57-65180).

However, in the above method, since the cells are cultured in a fixed amount of nutrients, the growth and proliferation of cells stops at a relatively low density.

(c) Structure of the Invention The present inventors overcame the drawbacks of the conventional methods as described above, and conducted research on a culture method that enables large-scale, high-density culture using the suspension culture method.
We have arrived at the present invention.

That is, the present invention allows nutrients and growth inhibitory substances necessary for cell culture to substantially permeate into the suspension liquid of a suspension type cell culture tank, but cells do not substantially permeate the suspension liquid (formed from wall membranes). A capillary bundle consisting of a large number of hollow fibers, both ends of which are fixed by an anchoring layer.
(1) The culture solution in the suspension solution is discharged from both ends or one end of the unit to the outside of the culture tank through a conduit through the hollow fiber wall membrane of the unit, and (ii) L After that, an aqueous liquid medium that does not adversely affect the cell culture is supplied from outside the culture tank to both ends or one end of the unit through a conduit, and the liquid medium flows into the suspension liquid through the wall membrane of the hollow fiber. This is a cell culture method characterized by alternately repeating discharge and inflow.

Furthermore, in the method of the present invention, at least two of the units are housed in the suspension-type cell culture tank, and each unit has the discharge and inflow intersecting with each other.
The cultivation can be carried out more advantageously by controlling the intervals between the discharge and inflow of each unit so that the culture solution in the suspension solution in the culture tank does not stop discharging. can.

Thus, according to the culture method of the present invention, at least one unit such as the capillary bundle is submerged in the suspension solution in a suspension-type culture tank, and the waste products 9 metabolites produced by the cells, The old culture solution containing other prebiotic-inhibiting substances flows from the suspension solution into the hollow fiber through the wall membrane of the hollow fiber, and is discharged from both ends or one end of the unit to the outside of the culture tank via a conduit. is stopped, and then an aqueous liquid medium that does not adversely affect the cell culture is supplied from outside the culture tank through conduits to both ends or one end of the unit, so that the nuclear liquid medium flows into the suspension liquid through the wall membrane of the hollow fiber.

Therefore, in the method according to the invention, by means of a unit submerged in suspension liquid.

The supply of the aqueous liquid medium into the suspension liquid through the many hollow fiber wall membranes provided therein and the discharge from the suspension liquid to the old culture liquid are alternately repeated, so that the hollow fiber wall membrane Pores & 1ffl Birds and other solids can clog without causing substantial wall area efficiency loss (advantageously, supply of aqueous liquid medium and draining of old culture medium can be carried out smoothly) , it becomes possible to culture cells at extremely high density.

In particular, in the method of the present invention, two or more units can be immersed in the suspension liquid separately or in a suitable combination, and two or more units can be easily connected in series or in parallel. It is possible to easily adapt the wall area of the hollow fiber according to changes in cell type and density, culture conditions, and culture scale.

Further, in the present invention, at least two units are used in combination, and each unit is operated so as to repeatedly supply the suspension liquid and drain the old culture liquid while being in the suspension liquid of the culture tank, and Make sure that at least one unit is constantly draining old culture fluid.

The intervals between the discharge and inflow of the liquid in each unit can be mutually controlled so that the discharge of the culture solution in the suspension liquid in the pickled culture tank does not stop.

The cell culture method of the present invention is used in a suspension type cell culture tank, and the suspension type means that the cells themselves are suspended in an aqueous medium, or the cells are supported on microcarriers (microcarriers) K. Refers to various suspension cultures in which cells are grown in suspension or in microcapsules. In particular, the present invention is advantageously used in a system in which the cells themselves are cultured while being suspended.

The cells cultured in the cell culture method of the present invention may be plant cells, animal cells, microbial cells, etc., or cells that have been modified artificially or by genetic manipulation.

The culture method of the present invention is particularly suitable for culturing animal cells.

In the suspension-type cell culture tank of the present invention, cells to be cultured are cultured in a suspended state in the culture solution. The culture solution consists of an aqueous medium that is essentially a substratum, to which additive components commonly used in cell culture, such as various inorganic salts, vitamin Q, enzyme traps, glucose, and amino acid 1 antibiotics, are added. Further, serum can be added to the culture solution, or a so-called serum-free medium that does not use serum can also be used as the culture solution.

In the method of the present invention, the "aqueous medium that does not adversely affect cell culture" that is supplied from outside the culture tank to the unit in the suspension solution through a conduit is mainly composed of reduced-solen water and contains the above-mentioned additive components. One or more can be used in addition. This aqueous medium may be a fresh culture medium containing all additional components necessary for the culture, and does not need to contain all of these components, some of which may be introduced into the suspension solution by other supply means. You can also. In addition, as the aqueous medium, the culture solution (
It is also possible to use part of the culture solution (hereinafter sometimes referred to as "old culture solution").

The hollow fiber capillary constituting the unit used in the method of the present invention may be made of various polymers, such as cellulose acetate, polysulfone, poly7-crylonitrile, and fluorine-based polymers. etc. are mentioned as preferable examples.

The wall membrane of a hollow fiber capillary has many micropores, and the wall membrane of the capillary allows nutrients and cell waste products, 9 metabolic products, etc. to pass through, but it does not allow cells or microcarriers to which they are attached to pass through. Microcapsules are provided with many pores that are sized to prevent penetration. When culturing cells in suspension,
Although the size of the pores depends on the size of the cells, it is generally appropriate for the average pore size to be 10 μm or less, preferably 8 μm or less. On the other hand, when cells are cultured by adhering to the surface of microcarriers (microcarriers) or when cells are cultured using microcapsules, the pores need to be large enough that they cannot pass through.

It is also desirable that the capillary wall membrane has the ability to permeate water to some extent. That is, it is advantageous for the wall membrane to have a water permeability coefficient (ld/W?·hr-mHg) of 10 or more, preferably 100 or more. On the other hand, there is no particular upper limit, but 20.000 or less, preferably 10.00
Desirably 0 or less.

Further, as for the wall membrane, compounds with a small molecular weight such as nutrients and cellular waste products 9 permeate, but compounds with a large molecular weight (for example, 1000 or more, preferably soo.

It is also possible to use an impermeable membrane, such as an ultrafiltration membrane.The cell culture method of the present invention will be explained in more detail below with reference to the accompanying drawings.

FIG. 1 shows an example of a schematic diagram for carrying out the cell culture method of the present invention.

FIG. 2-A, FIG. 2-B, and FIG. 2-C each show an example of a schematic diagram of a unit in the present invention.

Figure 1 shows the case where two units) 3-a and 3-b are operated as a pair to perform industrial culture. The aqueous medium containing nutrients etc. is passed from the supply tank 6 through the supply conduit 8 by the pump 7 to the switching valve 1.
2 or 14 to one unit. On the other hand, the old culture solution is discharged from the other unit and sent via the switching valve 13 or 15 through the discharge conduit 11 to the storage tank 9 for old culture solution by the pump 10.

Two units 3-a and 3-b shown in FIG.
In this case, both ends of a capillary bundle consisting of a large number of hollow fibers are fixed by a fixed layer. The upper part serves as a liquid inlet and outlet, and the lower part is sealed.

The culture tank 1 may be equipped with a stirring device g12 as shown in FIG. 1 so that the cells are effectively suspended in the suspension liquid.

It is not limited to a rotary stirring blade like stirring 2, but a magnetic stirrer may also be used. Furthermore, other stirring effects that can effectively suspend the cells in the suspension solution may be used.

Although the culture tank in FIG. 1 is shown as a single set of two units, the number of pairs is not limited to one, and a plurality of sets may also be used. Further, the units may not be used as a pair, but - independent units may be used, and the operations of draining the culture medium and supplying the aqueous liquid medium to these units may be repeated. It goes without saying that a large number of these independent units can be used.

Furthermore, the concentration of oxygen, carbon dioxide and nutrients in the culture tank,
Devices for measuring the pH value and maintaining the pH value are generally installed, but it goes without saying that the cell culture tank of the present invention may also be equipped with these devices. However, these accessories are omitted from FIG.

The new culture solution supplied to the culture tank of the present invention contains nutrients such as grapes, etc., and proteins.

An aqueous solution containing components necessary for cell culture, such as various amino acids, inorganic salts, and antibiotics, is used, but it may or may not further contain serum.

All of these components may be supplied as a culture solution through the unit, but some components may also be introduced into the suspension solution by other supply means. Generally, cell culture requires oxygen, carbon dioxide, etc., but these can be supplied dissolved in a new culture medium, and as shown in the first part, the gas supply pipe 17 is supplied through the valve 16. It may also be supplied directly to the suspension fluid.

2-A, 2-B, and 2-C also show examples of various forms of units used in the method of the present invention. In these figures, the arrow → indicates a conduit connected to the liquid inlet and outlet connected to either the switching valve 12, 13, 14 or 15 in the culture tank of FIG.

The unit shown in Fig. 2-A has basically the same form as the unit shown in Fig. 1), in which both ends of a capillary bundle consisting of a large number of hollow fibers are fixed by a fixing layer, and one end A common liquid inlet and outlet are provided at both ends, and the other end has a sealed structure. In Figure 2-B, both ends of the capillary bundle are fixed to the fixed layer, liquid inlets and outlets are provided at both ends, conduits are connected to each of these inlets and outlets, and these conduits are connected to form a common liquid inlet. It has a structure that forms a discharge port. Further, Fig. 2-C shows a form in which both ends of the capillary bundle are fixed by one common fixing layer,
It has a structure in which a common liquid inlet and outlet are provided at both ends. Each of the units described above is an example of a basic type, and other slight changes are naturally allowed. A plurality of these units can also be used in arbitrary combination.

During the process of the present invention, each unit is operated while alternately supplying an aqueous liquid medium (for example, fresh culture medium) and discharging old culture medium, the intervals of which are as follows:
Although it depends on the cell type, density, culture conditions, etc., it can generally be switched within a range of 1 minute to 5 minutes, preferably 5 minutes to 10 hours.

The method of the present invention can also be operated by submerging one unit in the suspension liquid and alternately repeating the supply and discharge of the liquid as described above, or by submerging two or more units to be operated in this manner and each May be operated independently.

However, it is generally preferable to use two or more units, in which case it is advantageous to operate the units in relation to one another.

When operating multiple units in conjunction with each other,
: Discharge of liquid from each unit so that the suspension of the suspension in the paddle will not stop discharging the culture liquid to the outside of the tank (so that the old culture liquid is constantly drained out of the tank) It is more desirable to mutually control the intervals between the inflow and the inflow. In addition, two units were submerged in the suspension liquid as a pair, and one of the units was operated so that the aqueous liquid medium was supplied into the suspension liquid, and the other unit was operated so that the old culture was discharged, and their flow was alternated. By operating the pair of units alternately in this way by switching the switching valve in the opposite direction, the supply of the aqueous liquid medium and the discharge of the old culture medium are carried out continuously at all times, and thus, Moreover, it becomes possible to easily maintain the liquid level of the suspension in the culture tank at a constant level.

When carrying out the method of the invention, the interval between the supply of the aqueous liquid medium and the discharge of the old culture medium in the unit can be arbitrarily determined. In general, draining the old culture solution can be carried out for a long time as long as the cells etc. do not cool the pores of the wall membrane of the hollow fibers and the amount of discharge decreases.
It is desirable to keep the suspension within 0 days; when the cell density in the suspension liquid is low, it lasts for a relatively long time; on the other hand, when the cell density is high, it tends to be busy for a short period of time.

On the other hand, supplying an aqueous liquid medium (eg fresh culture medium) generally takes less time than said draining. Generally, 1 minute or more is sufficient. The supply time is also influenced by the supply fi (area ratio); if the supply is busy, it can be shortened, and if the supply is busy, it may be necessary to lengthen it. Especially with a little pressure.

It is often advantageous to supply the aqueous liquid medium over a short period of time.

According to the culture method of the present invention as described above, in suspension culture, the supply of new culture solution and the discharge of old culture solution are carried out continuously or semi-continuously, so that a large number of cells can be grown at high density. It can be cultivated. In particular, in the present invention, even if cells or other solid matter temporarily clog the walls of the hollow fibers in the unit, the clogging will not occur for a long period of time, and therefore, the culture medium can be exchanged effectively. It is possible to perform extremely high-density culture.

Furthermore, the units of the present invention can be easily used by simply combining two or more units, in series or parallel, or by appropriately combining two or more units independently, while taking into account various factors such as temperature, equipment scale, cell type, and culture conditions. It has the advantage of being

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

Examples (Properties of 11 Units) Using a large number of hollow fibers formed from a polymer fiber made of cellulose acetate, an acrylic acid polymer, and a cellulose nitrate, as shown in Figure 2-A of the attached drawings, I made a unit with one side sealed.The inner diameter of each hollow fiber is 320/J and the outer diameter is 460μ.

The wall membrane had numerous micropores with an average pore size of 0.2μ. The effective length of the hollow fibers in the unit was 36 cm, and the total effective area (based on the outer diameter) of the hollow fibers was 80 csi. The water permeability of the hollow fiber used was 4J/m'-h.
It was r-Nishiki Hg.

(2) Shape of the culture tank; The structure is as shown in the attached Figure 1, and the internal volume is 56gm.
A glass container was used as a culture tank. This culture tank houses two units as shown in Figure 1.

In addition, a marine stirrer is installed and a carbon dioxide and oxygen supply conduit is inserted into the culture tank, which corresponds to 5 in Figure 1.

(3) New culture solution composition; The composition of the new culture medium used was as follows.

Units are KM (mmol) unless specified in %.
I'll go.

NaC1110, OmM KCI 5.0 Mg5Oa O, 55 MgCl, , 6H,0- ca (NOI)s, 4 uxo -CaC1,, 2
)1,0 0.74 NaH1PO4,2H,0- Na*llPO4,iza,o 3.41CuSO,,
5H103, OX 10-FeSO4, 7H@0 8
, OX 10-F e (N03) g , 9
Hlo -ZnSO4,7H1011,OX 10-'
A1an1n* o, o, ys Arginin*, HCl 2.76 Asparagine, Hlo 0+63 Ampara
tic acid 0.30・Cyst@In@, HC
l, Hlo 0.60Cya'Lotte-Glutamlc aeld O127Glutaml
na 6,84 Glycine 0.57 H1stldine, HCl, Hp 0.36Hydr
oxyproline 0.24Isol@ucine
1.20 Leuc1n@1.26 Lyslne, HCl 1.08 M@thto+htn* 0.33 Phsnylalanne O,45Proline
O・48 Setine O・81 Thr@one O,93 Tryptophan O00+1 Tyroalne O,48 Valjne O,LI p-Amlno benzo (c acid 3.7
Xl0-"Biotim 4.1 X 1G-' Pantoth@nat*-Ca 2.6 X 1G-
"Cholin chloride 8,8X10-"
Folie aell, 4. lX10-”Inom
ltol 2.6 X 10-'Nlac1mid
e 1.2 X 10-”Pyridoxal, HCl
4,9X1G-”Pyrldoxins, HCl2
．． 5 Xl0-”Rlboflavlne 5.6X1
0"Thlamin, HCl 4.7 x 10-"V
itamin B,, 2.5 x 10”Llpo
ic acid 2.5X10-4Glutathia
ne 1.6 X 10-”Glucose 19,0
0 Hypoxanthines 7.5 X10-”Put
resein*, 2HC12,5X 10-'Pyru
vate-Na 1. O Thymidlne 725 X 1G-”L%mol
@%e aala 7.5X1G-”Phenol r
@d 5.01! /LHEPE8 1.19.91/L NaHCO112,50WM Eltreptomyein 0.1 9/LP@n1
aillin G 10” U/LInmullne
7.5pl/l1lTransaphclln 2 μm
1/l Ethanolamins 10 μM/LNa
18.Os 2.5 X 10-'M/L (4) Culture method: A new culture solution was poured into the culture tank so that the pressure liquid volume was 300 d, and two units were inserted into it. Each unit is connected to a new culture solution supply tank and an old culture solution storage tank 9, as shown at 6 in FIG. 1, through a switching valve and a conduit, respectively. A pump is installed in the middle of each conduit, and the two units are operated by a switching valve so that one unit can drain the new culture solution out of the culture solution supply tank a in reverse flow. It is supposed to be done.

On the other hand, in the culture tank, CO, gas and 0. Gases were supplied into the suspension liquid, and their supply was controlled so that the pH within the system remained constant at 6.5-7 and the amount of dissolved oxygen remained constant at 3-4.

The mouse hybridoma 4C10B6 cell line, whose parent strain was mouse minipoma cannon p3ul, was added to the culture solution in the culture tank at a rate of 10,000 cells/dtft;b. 13cr) is a production type strain.

The suspension solution during culture was maintained at 27°C, and a Marinya stirrer was rotated at a speed of 60r%. After the culture was started and the cell density reached 1X10' cells/d, the two units were started to operate, and new culture solution was supplied and old culture solution was discharged. The interval between supply and discharge in one unit was approximately 12:00 S, and the supply of new culture solution to the culture tank in one day was approximately 300 days. However, after 5 days from the start of culture, the supply of this new culture solution was gradually increased to a maximum of 500 - per day.

(5) Culture results: After culturing for 9 days, the results were as shown below.

The final cell density was 281 G cells/ml when culturing was carried out under exactly the same conditions except that the unit of the present invention was not used.

[Brief explanation of drawings]

FIG. 1 shows an example of a schematic diagram for implementing the cell culture method of the present invention, and FIG. 2-A, FIG. 2-8, and FIG. An example of the form of the unit used in the cell culture method is shown. Figure 1

Claims (1)

[Scope of Claims] 1. The suspension liquid of a suspension type cell culture tank contains a wall membrane or a membrane that substantially permeates nutrients and growth-inhibiting substances necessary for cell culture, but does not substantially permeate cells. A capillary bundle consisting of a large number of formed hollow fibers, in which at least one unit σ fixed by a fixed layer is housed at both ends of the capillary bundle, and the culture solution in the middle suspension solution is transferred to the hollow fibers of the unit. After removing the wall membrane of the thread and draining it from both ends or one end of the unit to the outside of the culture tank through a conduit, drain an aqueous liquid ζ that does not have a negative effect on cell culture from the culture tank through the conduit. A cell culture method characterized in that the liquid medium is supplied to both ends or ends of the unit and flows into the suspension solution through the wall membrane of the hollow fiber, and the above-mentioned discharge and inflow are repeated alternately. 2. Suspension At least two such units are housed in the suspension solution of the type cell culture tank, and each unit is actively operated so that the discharge and inflow are repeated alternately, and the culture in the suspension solution in the culture tank is 2. The cell culture method according to claim 1, wherein the intervals between discharge and inflow of each busy unit are mutually controlled so that the discharge of the liquid does not stop.