JPH0265778A - Isolation of cell culture product and change of culture medium - Google Patents

Abstract

PURPOSE:To continuously and aseptically obtain the subject cell culture product using a small-sized device by isolating the product using an apparatus containing a bundle of hollow fibers capable of passing the product through without passing the cells through and separating the culture medium using another apparatus containing a bundle of other hollow fibers capable of passing the culture medium through without passing the cells and the product through. CONSTITUTION:Cells for culture and a culture medium are supplied through a supply unit 10 into a vessel 8 for the culture medium of a cell culture incubator 7 and the cells are cultured. The product is isolated using an apparatus 1 containing a bundle of hollow fibers capable of passing the product through without passing the cells through supported inside the vessel by a supporting unit 13 and taken out from a takeoff line 23 and the culture medium is taken out from another takeoff line 24 using an unit 9 supported inside the vessel by a supporting unit 14 containing a bundle of other hollow fibers capable of passing the culture medium through without passing the cells or the product through and changed with a fresh culture medium, thus efficiently carrying out takeoff of the cell culture product and change of the culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for removing products obtained by culturing cells and replacing a medium.

[Problems to be solved by conventional techniques and inventions] In recent years, with the progress of biotechnology, various devices for culturing and utilizing microorganisms, cells, etc. have been proposed.

For example, as a device for culturing cells, there are tank-type reactors such as semi-batch reactors, which are partially continuous reactors that require batch operation, and continuous tank heavy reactors, which are improved from quarter-part operation to continuous operation. Front-type reactors suitable for continuous operation have been proposed and used.

The above improvements have some effect in making batch operations semi-continuous or continuous, but in both cases, it is difficult to separate cells, culture medium, and other necessary components (e.g., serum, physiologically active substances such as lymphokines, etc.). This is a method in which a desired cultured cell or product is obtained by stirring the system in one system, and is not a method in which the culture medium is replaced and cultured under a preferable condition.

Since cultured cells and useful products exist together with a large amount of medium, it is not easy to separate and purify the desired product, and it is difficult to increase the concentration of cultured cells and products. An important issue is how to reduce the amount of culture medium that interferes with the extraction operation of living organisms.

[Means to solve the problem]

The present invention has been made in order to improve the drawbacks of the conventional methods as described above. Removal of the culture medium (1) is carried out using a device (■) comprising a fiber bundle (hereinafter also referred to as the hollow fiber bundle device of (I)), using a hollow fiber bundle that does not pass through the cells and products but allows the culture medium to pass through. (N) (hereinafter referred to as (H)
This invention relates to a method for removing cell culture products and replacing a medium, which is carried out using a hollow fiber bundle device (also referred to as a hollow fiber bundle device).

[For production]

In the present invention, the culture medium is removed by a hollow fiber bundle device (cap) that does not permeate cells and products such as useful metabolites, but allows the medium to pass through, and the culture system is maintained in a favorable state.
The product is removed from the culture system in which the product concentration is increased by means of a hollow fiber bundle device (I) which permeates the product but not the cells. This operation is performed continuously under aseptic conditions.

Furthermore, the hollow fiber bundle devices (I) and (II) have a large surface area effective for permeation compared to the size of the device, and can be efficiently taken out with a small device.

〔Example〕

In the present invention, when taking out products obtained by culturing cells, the medium is taken out by the hollow fiber bundle device (II) and the culture system is maintained in a favorable state, and the hollow fiber bundle device (I) The product is removed.

fI)'s hollow fiber bundle device will be explained based on the device shown in FIG. 1, which is an example thereof.

(The hollow fiber bundle device (I) of I1 does not penetrate cells, but
This is a device for permeating useful products produced by culture and taking out the products outside the culture system.Both ends of the hollow fiber bundle (2) are supported by support means (3), (3゛), and a cap (6) whose lower end is open and which has liquid passages (5), (5') in the openings (4), (4');
6°) is the device that is crowned.

The hollow fiber bundle in the apparatus shown in Fig. 1 is an example in which the upper end is open, and the hollow fiber bundle apparatus can be easily brimmed before use, and can be formed vertically symmetrically, making it easy to manufacture. does not need to be open, but may be closed by a support means (3) or a cap (4). However, if the upper end is directly blocked by the support means (3) etc., it is difficult to brim the hollow fiber bundle device before use, and if brimming is not done, air block will occur and the liquid recovery efficiency will be reduced. .

The hollow fibers constituting the hollow fiber bundle ('2J) are not particularly limited as long as they do not permeate cells but permeate products; (For example, if the molecular weight of the product is 100,000 to 1 million, such as immunoglobulin M (IgM) or fibrinogen, the average pore size is about 500 to 5,000, and if the molecular weight is 1,000,000 to 1,000,000 like albumin.
In the case of 10,000 to 100,000, the average pore size is 50 to 500.
Hollow fibers (about the size of a human body) are preferably used.

There are no particular limitations on the material constituting the hollow fibers, but
Hydrophilic materials 1), such as for example regenerated cellulose, are preferred in order to allow the products present in the medium to permeate. For example, cellulose triacetate, cellulose diacetate,
In the case of hollow fibers made of non-hydrophilic materials such as polyethylene, polypropylene, polyester, polyamide, polytetrafluoroethylene, and polysulfone, it is necessary to use hollow fibers whose surfaces are made hydrophilic.

Usually about 300 to 600 hollow fibers are bundled and both ends are tied together.
For example, the hollow fiber bundle (2) is formed by potting with urethane resin or the like to form potting portions that serve as support means (3) and (3°).

The cross-sectional shape, length, etc. of the bundled hollow fibers may be selected as appropriate, but it is preferable to make the contact area with the culture medium large, and those having a circular cross-sectional shape are usually adopted.

The hollow fiber bundle device (II) is impermeable to cells and products, but permeates and drains the medium to maintain the medium in a desirable state.

The structure of the hollow fiber bundle device (if) may be the same as the hollow fiber bundle device (I), and the difference between these two devices is that the substances to be transmitted are different, that is, in the case of the hollow fiber bundle device of m. In contrast, in the case of the hollow fiber bundle device (II), the product does not permeate, but mainly permeates the medium. Therefore, the average pore size of the hollow fibers constituting these devices is different (the average pore size of the hollow fibers in the hollow fiber bundle device of Ill.
When 50 hollow fibers are used, serum and cell activating substances such as lymphokines do not pass through, but the medium passes through, so serum and cell activating substances can be saved.

Next, the method of the present invention will be explained based on the apparatus shown in FIG. 2, which is an example of a culture apparatus used in the method of the present invention.

In Figure 2, (7) is a cell culture device, (8) is a culture medium container that is a container for culture solution containing cells, serum, lymphokines, etc., and (I) is a cell culture device that does not permeate cells, but does permeate products. A hollow fiber bundle device comprising a hollow fiber bundle (9)
) is a hollow fiber bundle device comprising a hollow fiber bundle that does not permeate cells and products but allows permeation of a medium; ω) is a medium supply means for supplying a medium, etc.; 01) is a degassing means having an air filter side; D, 04) is a holding means having liquid passages communicating with the hollow fiber bundle devices (I) and (9), respectively, and its upper end is closed so as to be openable by a cock or the like.

The medium and the like are supplied by medium supply means M. At this time, it is preferable to perform this with the gas venting means 011 open in order to vent the air in the culture medium container (8).

The cells to be cultured may be supplied by a medium supply means, but a separate medium supply means is suitable for continuous operation in which the culture medium is continuously supplied and the products and the medium are continuously taken out. preferred.

Cells to be cultured, serum, a cell activating substance such as lymphokine, a medium, and the like are supplied to a culture medium container (8), and culture is performed under conditions such as temperature depending on the culture conditions of the cells.

At this time, gas is supplied from a gas supply means (not shown) using an air pump or the like through an air filter of about 0.2 mm, gas exchange is performed, and the exhaust gas is In addition, the medium and the like are supplied from the medium supply means ω.

For example, as shown in FIG. 3, the useful products produced by the culture pass through the pores of the hollow fibers Oe constituting the hollow fiber bundle device (+1), and the nutrients (g) in the medium, For example, if the cell activating substance 09 is more likely to permeate the hollow fiber than the product, it is taken out from the hollow fiber bundle device (I) together with the cell activating substance plane.

In Fig. 3, the cells are cells, and the cells are serum.

At this time, as shown in FIG. 4, the medium containing serum (2Il and cell activating substance Og) is transferred from the hollow Kento device (II) by the hollow fibers n constituting the hollow fiber bundle device (11). taken out.

When taking out the product using the hollow Kento device (m) and discharging the culture medium by the hollow Kento device (ff), if the negative pressure due to the siphon effect is insufficient, It goes without saying that means such as a pump may be provided.

Although a stirring means is not necessarily required for the apparatus used in the method of the present invention, if a stirring means is provided to stir the medium, it becomes easier to maintain the medium in a constant state, and it becomes easier to culture the cells. In this case, it is preferable to use a device having a frame with a gap, for example, as the hollow Kento device (I) and the hollow Kento device (fit).

Since the frame is for fixing the hollow fiber bundle (21) which is likely to cause problems due to shaking due to stirring, it is preferable that the frame is one that allows products in the culture medium to easily pass through. There are no particular limitations on shape, size, etc. as long as the purpose can be achieved.

Therefore, the shape of the void existing in the frame may be, for example, a wide, long strip-like void, a slit-like shape, a lattice-like shape, a circular to an elliptical shape, or the like.

Further, the porosity is preferably as large as possible as long as the hollow fiber bundle (2) can be fixed.

Although FIG. 2 does not show any means for monitoring the condition of the medium during culture, it is a matter of course that culture can be carried out while monitoring the condition from time to time.

Cells to be cultured by the method of the present invention include, for example, lymphocytes, bone marrow cells, macrophages, etc. Cell activators used at this time include, for example, lymphokines and interferons, and the culture medium includes:
Examples include fetal bovine serum medium, serum-free medium, and human serum medium. Examples of the products include physiologically active substances such as immunoglobulin fibrinogen, albumin, lymphokine, insulin, interferon, urokinase, growth hormone, and monoclonal antibodies.

Next, the method of the present invention will be explained based on examples.

Example 1 An apparatus substantially similar to that shown in FIG. 2 was used.

The culture medium container (8) in Fig. 2 is made of hard glass and has a capacity of
i3300ml, diameter approx. 150sm x height approx. 190+n
The cylindrical container and (I) hollow Kento device are made by bundling 300 hollow fibers made of hydrophilized cellulose triacetate with an average pore size of 0.1 and botting them so that the effective length is 120 am. (Total surface area: 250 cj), both ends of the hollow fiber are open, and each end is covered with a cap having a liquid passage. (Ill's Hollow Kento Equipment is made by bundling 300 hollow fibers made of hydrophilic cellulose triacetate with an average pore size of 30, and has an effective length of 1.
Both ends of the hollow fibers are open and covered with caps containing liquid passages. Then, on the lid body that shuts off the culture medium container (8) from the outside, the hollow Kento device (I) (I), (9), the medium supply means and the
．． This device is equipped with a 2Ja air filter (gas venting means 01 having I21). Note that (I) above,
(II) The hollow Kento devices (I) and (9) were held by respective holding means of 0.0Φ, and the upper ends were closed. Further, an air pump (not shown) was attached via an air filter (0,2 addition) to supply gas.

With the gas venting means 01) open, RPMI-1 was added as a medium.
640 (manufactured by Ajinomoto ■) 10100Q was supplied, and then 110 ml of cell suspension consisting of 106 lymphocytes/ml x 100 ml, 10 ml of serum, and 200 units of lymphokine was supplied to the medium container (8). Thereafter, culture was performed at 37°C. During this time, the culture medium is supplied from the medium supply means at a rate of 1ml/1n, and air containing 5% by volume of CO2 is supplied using an air pump at a flow rate of 1ml/1n.
The cells were supplied at a rate of 0 ml/min and cultured for 20 days.

During culture, the pi and partial pressure of carbon dioxide (PCO) of the medium are adjusted every day.
z), 7.2≦pl+≦7.3.36mm1
It was confirmed that 1g≦PCO2≦40mLI1g.

In addition, a liquid containing the product immunoglobulin N1 was collected from the hollow fiber bundle device (I) at a rate of 0.1 ml/min, and 0.9 ml of the culture medium was collected from the hollow fiber bundle device (n).
The fluid was drained at a rate of /akin.

〔Effect of the invention〕

According to the method of the present invention, a product is produced aseptically and continuously, and a liquid containing the product can be removed using a compact device. Furthermore, cell activating substances added to the culture medium can be saved.

(Main symbols in the drawing) (I) = (I) Hollow fiber bundle device (2) of (I), Hollow fiber bundle (9): Hollow fiber bundle device of 劃): Product head: Cell

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of an example of the H hollow fiber bundle device used in the method of the present invention, FIG. 2 is an explanatory diagram of an example of the device used in the method of the present invention, and FIG. 3 is a +11
FIG. 4 is an explanatory diagram showing a state in which a product is taken out by a hollow fiber bundle device. FIG. 'A11 Diagram 2nd port

Claims (1)

[Claims] 1. When cells are cultured and their products are obtained, the products are taken out using a device (I) comprising a hollow fiber bundle that does not pass through the cells but allows the products to pass through. A method for removing a cell culture product and exchanging a medium, characterized in that the medium is removed using a device (II) comprising a hollow fiber bundle that does not pass through cells and products but allows the medium to pass through. 2. The hollow fiber bundle in the device (I) is a hollow fiber made of regenerated cellulose having an average pore size of 50 Å to 0.5 μm, or hydrophilized cellulose triacetate,
2. The method of claim 1, comprising hollow fibers made of cellulose diacetate, polyethylene, polypropylene, polyester, polyamide, polytetrafluoroethylene or polysulfone. 3 The hollow fiber bundle in the device (II) is made of regenerated cellulose hollow fibers having an average pore size of 30 to 50 Å, or hydrophilized cellulose triacetate, cellulose diacetate, polyethylene, polypropylene, polyester, polyamide, polytetrafluoroethylene. The method according to claim 1, comprising hollow fibers made of polysulfone or polysulfone.