JPH0797982B2 - Cell culture device - Google Patents

Description

TECHNICAL FIELD The present invention relates to a cell culture device, and in particular, efficiently supplies nutrients to cells and skillfully removes unwanted substances by-produced while concentrating useful useful substances. The present invention relates to a hollow fiber type cell culture device which is suitable for achieving high density continuous cell culture by achieving uniform environment in the system.

[Prior Art] High-density cell mass culture is an essential technology for industrial-scale production of monoclonal antibodies, lymphokines, and other useful physiologically active substances.

Although classical cell culture has been performed at the laboratory level using culture bottles and the like, it is industrially advantageous if cell culture can be performed at a higher density and in a large amount. Various methods and devices have been developed.

The culturing methods in these methods and devices are roughly classified into floating (non-adhesive) cell cultures and adherent (wall-adhesive) cell cultures according to the characteristics of the cells to be cultivated. It is important to maintain the suspension state and efficient substance exchange, while in adherent cell culture, efficiently increase the number of adherent cells per unit volume and efficiently exchange substances such as nutrients, waste products and gas. It will be a point.

Regardless of which culture method is adopted, since substance exchange is an important point, cell culture using hollow fibers or capillaries having selective separation has attracted attention, and for example, substance exchange performed using hollow fibers is , Which is similar to the fact that cells in vivo obtain nutrients from blood through blood vessels and excretes metabolites, and is regarded as an extremely promising in vitro cell culture method, and various culture devices will be developed. Has arrived. Among them, a method of flowing a liquid medium (culture solution) into the hollow fibers to attach cells to the outer surface of the hollow fibers to proliferate, or flowing a gas containing oxygen into the hollow fibers to exist in the liquid medium on the outer surface of the hollow fibers. Various improvements such as positive gas exchange of cells are underway. Cell culture techniques using such hollow fibers have hitherto been disclosed in JP-A-49-41579, JP-A-50-36684, and JP-A-52-125688.
59-175877, 61-25477, published patent 62-50035
No. 6 and No. 62-500357 are disclosed.

[Problems to be Solved by the Invention] As limiting factors for high-density culture of cells, accumulation of growth inhibitory substances (eg, metabolites such as lactate and ammonium ions) in the system, essential nutrients (eg, glucose, essential) Depletion of amino acids, vitamins, etc., depletion of the amount of dissolved oxygen, and a sharp drop in pH.

The conventional hollow fiber cell culture device was developed for the purpose of completely eliminating such limiting factors, but it was difficult to say that it was a technology that established high-density cell culture. . In particular, the environment in the culture device is not completely uniform, and the growth of cells is partially insufficient. As a result, the cell density of the entire culture device is not as high as expected.

The present invention has been made to solve these technical problems, and an object thereof is to make the environment in the cell culture device as uniform as possible, and thereby to perform cell culture at high density. It is to provide a hollow fiber type cell culture device that can be used.

[Means for Solving Problems] According to the present invention capable of achieving the above object, the culture solution is supplied under pressure to a plurality of hollow fibers having selective permeability, and a part of the hollow fibers is In a cell culture device configured to permeate to the outer peripheral side and perform cell culture in a cell culture space formed in the outer peripheral region, a culture solution circulation for circulating the culture solution supplied in the hollow fibers under pressure. A system, a culture solution containing a low molecular weight substance and a macromolecule useful substance generated in the cell culture space, a production liquid circulation system for circulating through a membrane separation device for separating the low molecular weight substance, Further, the cell culture device has a gist in that it is provided with a pressure adjusting means for maintaining a flow rate balance between the culture solution circulation system and the product solution circulation system.

[Operation] In the present invention, the basic constitution is to prevent the depletion of nutrients, dissolved oxygen and the like in the cell culture space by supplying the culture solution into the hollow fibers under pressure. In this case, it is possible to supply the culture solution under the optimum culture condition by adjusting the composition of the nutrient source supplied in the hollow fiber, the amount of dissolved oxygen, and the like in advance.

By adopting the configuration of the culture solution circulation system in which the culture solution supplied into the hollow fibers is circulated under pressure, the culture solution can be uniformly supplied throughout the cell culture space. In order to more effectively achieve this purpose, a gas exchanger, preferably a hollow fiber type artificial lung, is provided in the culture solution circulation system to control the amount of dissolved oxygen or the amount of dissolved carbon dioxide gas, and a fresh culture solution or a base is added. It is recommended to provide a supply line to supplement the culture medium that has permeated the hollow fibers and adjust the pH.

The type of cells in the cell culture space is not limited at all, and in the case of adherent cells, a method of attaching the cells on the outer surface of the hollow fiber or a method of enclosing the cells in a polymer gel matrix such as collagen ( Japanese Patent Application Sho 62-65363
No.) and the like, and on the other hand, for doubling the number of floating cells, there may be mentioned a method of confining the cells in the polymer matrix, a method of culturing in a liquid suspension state, and the like.

The culture solution in the cell culture space is guided to the membrane separation device in a state of containing the generated low-molecular unnecessary substance and high-molecular useful substance. The culture fluid guided to the membrane separator is circulated again in the cell culture space after removing low molecular weight unnecessary substances such as lactate and ammonium ions which are cell metabolites, which are growth inhibitors, (product solution circulation). system). The pH and the amount of dissolved oxygen of the circulating culture solution are adjusted in the product circulation system.

By circulating the culture solution in the cell culture space through the membrane separator in this manner, cell metabolites can be rapidly removed from the vicinity of the cells, and the environment in the cell culture space can be made uniform. it can. This also results in partial or total pH in the cell culture space due to accumulation of metabolites.
It is possible to prevent a sharp decrease in Furthermore, by selectively separating and removing unnecessary substances of low molecular weight with a membrane separator, it is possible to reduce the concentration of the growth inhibitor in the culture solution and to concentrate the useful polymeric substances produced by the cells. Then, the culture solution in which the polymer useful substance is concentrated is taken out of the system at a constant rate, and the movement of the culture solution in the cell culture device is stabilized by controlling the flow rate of the culture solution permeating through the hollow fibers. Further, continuous culture in the cell culture device becomes possible, and it becomes possible to stably collect a high-concentration useful substance for a long period of time.

In the membrane separation device, it is desirable that only low-molecular weight substances be selectively removed, and the molecular weight cut-off thereof should be at least in the range in which a high-molecular useful substance does not permeate, for example, about 1000 to 200,000, preferably 3000 to It is appropriate to set it to about 30,000. On the other hand, the molecular weight cutoff in the hollow fiber may be set within a range in which the useful substance does not permeate,
It may be greater than or equal to the molecular weight cut-off of the membrane separator.

The material of the hollow fiber in the device of the present invention is not particularly limited, and examples thereof include an organic polymer, an inorganic porous material or a metal porous material. The inner diameter is 10 to 1000 μm,
The film thickness may be 2 to 500 μm, and the inner diameter is preferably about 50 to 500 μm. Examples of the organic polymer material include cellulose acetate, cellulose triacetate, cellulose ester, polysulfone, polyolefin, polyfluorocarbon, and polysiloxane.

The circulation flow rate in the culture medium circulation system is 1000 cm / min or less in terms of linear velocity, particularly preferably 300 cm / min or less, and 1000 cm / min.
If it exceeds the limit, the pressure in the hollow fiber becomes too large, and the amount of permeation into the cell culture space increases, which is not preferable.

On the other hand, the circulation flow rate in the product circulation system is 0.1 to 1 in linear velocity.
It is preferably 000 cm / min, particularly about 1 to 300 cm / min. Then
If it is less than 0.1 cm / min, replacement of the liquid cannot be achieved, and the concentration of metabolites in the vicinity of the cell will be too high, and cell growth will be inhibited, and if it exceeds 1000 cm / min, the destruction and death of the cell will be caused by the effect of shearing force. An unfavorable phenomenon occurs.

The movement amount of the culture solution that permeates the hollow fiber is, for example,
As shown in the figure (Example drawing), a chamber 7 is provided in the culture solution circulation system, a gas pressure is applied to the culture solution 5 in the chamber 7, and the pressure is adjusted to control the pressure in the hollow fiber 1. The amount of movement is controlled by the control. Then, by controlling the liquid surface of the culture solution 5 in the chamber 7 and the liquid surface of the culture solution (product solution) in the chamber 19 provided in the product solution circulation system to be constant, respectively, The flow of the culture solution is stable, and long-term continuous culture is possible.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are not of a nature limiting the present invention.
It is within the technical scope of the present invention to add various design changes to the purpose of the following description.

[Embodiment] FIG. 1 is a schematic explanatory view showing an embodiment of the present invention.

The cell culture device according to the present invention is basically a cell culture device 2
And a membrane separation device 15. And the cell culture device 2
Comprises a plurality of hollow fibers 1 having selective permeability contained in a container 3, a cell culture space 4 is formed between the outer peripheral region of the hollow fiber 1 and the container 3, and the cell culture space 4 is formed in the space 4. Are filled with cells to be cultured.

Inside the hollow fiber 1, a culture solution 5 is fed by a liquid feed pump 6.
Is supplied. The culture solution 5 is stored in the chamber 7, and a propulsive force is given by the pressure applied by the air pressure supply device 8, and a part of the culture solution 5 permeates into the cell culture space 4. The remaining culture solution 5 that did not permeate into the cell culture space 4 is guided to the gas exchanger 9 through the line 20, and the concentration of oxygen and carbon dioxide gas is adjusted by the gas exchanger 9, and then fresh culture is performed. Liquid 10 and optimum pH adjusting base 11 (for example, 0.5N-NaOH) are added and returned to chamber 7 again (culture liquid circulation system). Further, the line 20 is provided with a pH sensor 12a and a dissolved oxygen amount detection sensor 13a, and the pH and the dissolved oxygen amount of the culture solution 5 passing through the line 20 are measured.

On the other hand, the culture medium 5 in the cell culture space 4 contains the generated low molecular weight substance and macromolecule useful substance (hereinafter referred to as the generated liquid 5a), and is temporarily stored in the chamber 19 and then the liquid delivery pump 14 is used for the membrane separation device. Supplied to 15. Then, in the membrane separation device 15, the separation membrane 16 is generated by the dynamic pressure of the liquid feed pump 14.
Low molecular weight metabolites such as lactic acid and ammonium ions in the production liquid 5a are separated through the (hollow fiber membrane in this example) and taken out to the outside of the system 17. The structure of the separation membrane 16 is not limited to the illustrated hollow fiber membrane, and may be a commonly used flat membrane or tubular ultrafiltration membrane or reverse osmosis membrane.

The product liquid 5a that did not permeate through the separation membrane 16 was then added to the line.
It is returned to the cell culture space 4 again through 21 (product solution circulation system), the inside of the cell culture space 4 is appropriately stirred, and the culture solution 5 (or the product solution 5a) near the cells and metabolites are uniformly dispersed. To help. Since the polymer useful substance in the product liquid 5a cannot permeate the separation membrane 16, it is concentrated as it passes through the product liquid circulation system described above. This macromolecular useful substance can be continuously taken out to a high concentration by recovering the product liquid 5a at a constant flow rate by the liquid feed pump 18 provided in the line 21, and the metabolites near the cells can be appropriately removed. In combination with this, the environment in the cell culture space 4 can be maintained uniform, and cells can be cultured at high density for a long time. Further, the line 21 is provided with a pH sensor 12b and a dissolved oxygen amount detection sensor 13b, and the pH and the dissolved oxygen amount of the product liquid passing through the line 21 are measured.

In the embodiment shown in FIG. 1, the pH sensors 12a and 12b and the dissolved oxygen amount detection sensors 13a and 13b are provided in both the culture solution circulation system and the product solution circulation system, but they are provided in either circulation system. It may be configured.

In this example, the hollow fiber 1 was made of cellulose acetate and had a molecular weight cutoff of 22,000, and the separation membrane 16 was made of cellulose acetate and had a molecular weight cutoff of 5000.

On the other hand, in order to stabilize the flow of the culture solution 5 and the product solution 5a in the cell culture device, liquid level gauges 22a and 22b are provided in the chamber 7 of the culture solution circulation system and the chamber 19 of the product solution circulation system, respectively. The pressure in the chamber 7 may be controlled by the air pressure supply device 8 so that the liquid levels in the chambers 7 and 19 of the chamber 7 and 19 are constant. The balance of the flow rate of the product liquid circulation system can be maintained. The same effect can be achieved by providing the air pressure supply device 8 also in the chamber 19 and adjusting the pressure in the chamber 7 and the pressure in the chamber 19. However, in such a case, it is necessary to adjust the pressure inside the chamber 7 to be higher than the pressure inside the chamber 19. Further, in order to achieve the object of the present invention, it is preferable that the liquid feed pumps 6 and 14 have a pulsation that operates in synchronization, and also considering the necessity of maintaining an aseptic state for a long period of time. As the liquid feed pumps 6 and 14, it is optimal to use bellows pumps having excellent airtightness.

FIG. 2 is a schematic explanatory view of another embodiment of the present invention. The basic structure is similar to the structure shown in FIG. 1 and the corresponding portions are denoted by the same reference numerals and redundant description will be given. Avoid In addition, in this embodiment, a line 23 is provided for returning a part of the product liquid 5a taken out of the system 17 to the outside 17 to the line 21. That is, in the low molecular weight substances separated by the membrane separator 15, in addition to growth inhibitory substances such as lactic acid and ammonium ions, nutrient sources useful for cell growth such as inorganic salts and amino acids are also included, The configuration shown in FIG. 2 is adopted in order to utilize these nutrient sources as much as possible. However, it goes without saying that the returned amount in this case needs to be such that the growth inhibitor contained therein does not inhibit the growth of cells.

From the same viewpoint as the configuration shown in FIG. 2, for example, the configuration shown in FIG. 3 can also be adopted. That is, in the configuration shown in FIG. 3, the product liquid 5a taken out from the membrane separation device 15 to the outside 17 of the system.
A line 24 for returning a part of the above to the line 20 of the culture solution circulation system is provided to achieve the same effect as the configuration shown in FIG.

FIG. 4 is a schematic explanatory view of still another embodiment of the present invention.
In this example, in order to supplement the nutrient sources such as the inorganic salts and amino acids taken out together with the growth inhibitor in the outside system 17, an aqueous solution 25 containing the inorganic salts and amino acids was separately prepared, and this aqueous solution 25 was supplied to the line 26. It is supplied to the line 21 through the above.

[Effects of the Invention] As described above, according to the present invention, by adopting the configuration described above, the environment in the cell culture device can be maintained as uniform as possible, and high-density continuous culture of cells becomes possible, It has become possible to obtain a high concentration of useful substances over a long period of time.

[Brief description of drawings]

1 to 4 are schematic explanatory views showing various embodiments of the present invention. 1 ... Hollow fiber, 2 ... Cell culture vessel 3 ... Vessel, 4 ... Cell culture space 5 ... Culture solution, 5a ... Product solution 6,14, 18 ... Delivery pump 7.19 ... Chamber, 8 ...... Air pressure supply device 9 ...... Gas exchanger, 15 ...... Membrane separation device

Claims (1)

[Claims] 1. A culture solution is supplied under pressure into a plurality of hollow fibers having selective permeability to allow a part thereof to permeate to the outer peripheral side of the hollow fibers, and in a cell culture space formed in the outer peripheral region. In a cell culture device configured to perform cell culture, a culture fluid circulation system for circulating the culture fluid supplied into the hollow fibers under pressure, and a low molecular weight substance and a polymer produced in the cell culture space. Equipped with a product solution circulation system that circulates a culture solution containing a useful substance through a membrane separation device for separating the low-molecular substances, and further maintains the flow rate balance between the culture solution circulation system and the product solution circulation system. A cell culturing apparatus, characterized in that it is provided with a pressure adjusting means.