JPH0634699B2 - Animal cell culture method and device - Google Patents

Description

TECHNICAL FIELD The present invention relates to a culturing method for culturing animal cells in high density and in a large amount, and a culturing tank used therefor.

[Prior art]

In recent years, advances in biotechnology have led to the development of technologies for producing pharmaceutically useful substances by genetically improving microorganisms or animal cells. Examples are interferon, human growth hormone, and insulin. However, when a microorganism is genetically engineered to obtain a target product (useful protein), the protein obtained therefrom has no sugar chain portion, and the whole structure differs from the natural type produced by animal cells. It is believed that proteins produced in animal cells will become the mainstream in the future due to their pharmacological effects.

On the other hand, since useful production substances brought about by the progress of genetic engineering improvement techniques for animal cells are in minute quantities, from the standpoint of producing such substances in large quantities, establishment of a technique for mass-culturing animal cells is desired. ing. As a conventional animal cell culture device, roller bottle, spiral film type, tube type, hollow fiber type, microcarrier type, air lift type, etc. have been developed including laboratory scale, and in industrial scale, floating The air-lift type and the holofiber type are suitable for culturing, and the microcarrier type is suitable for culturing animal cells having strong adhesion dependence.
In addition, a cell (tissue) compatible material as a cell culture bed for culturing adhesion-dependent animal cells or tissues has been studied, and it has been reported that polypeptides such as gelatin and collagen bring excellent effects. Since the polypeptide has high biocompatibility with animal cells, the experimental fact that by using this as an animal cell culture bed substrate, the cell adhesion is good, the growth rate is large, and high cell density culture can be performed. Known as. As a polypeptide-based culture bed, microcarriers in which gelatin is coated on a bead-shaped support of a dextran compound are sold. This microcarrier has a large surface area of about 20 cm ² per 1 m of liquid medium depending on the amount charged, and is said to be suitable for cultivation on an industrial scale.

However, the manufacturing method of this microcarrier is extremely complicated and the manufacturing cost is high, and in addition, in order to maintain the suspended state in the culture solution, the difference in specific gravity from the culture solution is taken into consideration, and the stirring is controlled. It is necessary and its handling is not easy. Furthermore, in the culture of adhesion-dependent animal cells, it is preferable to place it in a stationary state rather than a floating state,
The shearing force due to stirring also causes inconvenience to the culture.

〔Purpose〕

Here, the present invention provides a method for efficiently culturing animal cells by immobilizing a culture bed capable of ensuring a high specific surface area polypeptide surface in a culture tank, and for mass-culturing animal cells on an industrial scale. The purpose is to provide a suitable culture tank.

〔Constitution〕

According to the present invention, as a first invention, in a method for culturing animal cells under oxygen supply, a non-woven fabric having a polypeptide surface and a thickness of 0.03 to 0.3 mm and a porosity of 90 to 98% is used as a fixed bed. Adhesion-dependent animal cells can be efficiently cultivated by using as a culture bed and culturing while circulating the liquid medium through the culture bed, and by circulating the liquid medium circulating through the culture bed to the culture bed. An animal cell culture method is provided, and as a second invention, a porosity of 90 to 98 consisting of fibers having a polypeptide surface and a thickness of 0.03 to 0.3 mm.
% Non-woven fabric and an adhesion-dependent animal cell provided with a support member for supporting and fixing the non-woven fabric can be efficiently cultivated, and an animal cell culture tank that is easy to increase in size is provided.

In the present invention, a culture bed having a high specific surface area polypeptide surface and capable of maintaining its structure throughout the culture period is used. According to the research conducted by the present inventors, it has been found that such a culture bed can be obtained by constructing using a non-woven fabric. What is important in the present invention is to use a culture bed in which the surface area ratio of the polypeptide surface to the culture bed volume is large in order to efficiently and densely culture the adhesion-dependent animal cells.

Examples of fibers constituting the non-woven fabric used in the present invention include synthetic fibers made of synthetic resins such as nylon, polyester, acrylic resin, polyvinyl chloride, polyvinyl alcohol, polyethylene, polypropylene, polyurethane and polyvinylidene chloride, and stainless steel. Various fibers such as metal fibers, rayon fibers, glass fibers and carbon fibers are used alone or in the form of a mixture of two or more kinds. The thickness of the fiber is 0.03 to 0.3 mm. If the fibers are thin,
Although the surface area to volume ratio can be increased, the strength as a liquid culture medium support decreases, and when the fiber is thick, the strength as a liquid culture medium support is high, but the surface area to volume ratio becomes small. The non-woven fabric has a porosity of 90 to 98%. When the porosity is small, the surface area to volume ratio is large, but the culture bed is likely to be clogged, and when the porosity is large, the surface area ratio is small and the strength of the culture bed is low. In particular, when the porosity of the non-woven fabric is lower than 90%, the force applied to the non-woven fabric when passing the liquid medium through the non-woven fabric becomes large and the non-woven fabric is easily damaged, which is not preferable. On the other hand, when it exceeds 98%, the fiber density in the non-woven fabric becomes small, and the specific surface area (cm ² / cm ³ ) of the fiber becomes too low to obtain an efficient culture bed. The thickness of the non-woven fabric is in the range of several millimeters to several hundred millimeters, preferably 3 to 50 mm, and the area and the number of laminated layers may be appropriately determined in consideration of the size of the culture tank.

Next, the nonwoven fabric is surface-treated with a polypeptide,
The surface of the polypeptide is obtained by forming a polypeptide film on the fibers constituting the non-woven fabric and, in some cases, forming a film of the polypeptide between the fibers. In this case, as the polypeptide, a conventionally known one, preferably, collagen or gelatin is used, and collagen,
Gelatin includes these derivatives. After the surface treatment of the non-woven fabric with the polypeptide, the strength of the polypeptide surface can be increased by cross-linking with a cross-linking agent such as glutaraldehyde, ultraviolet rays, or γ rays. That is, by using a fiber structure, a high specific surface area and strength for maintaining its structure throughout the culture period can be obtained, and by surface-treating the fiber structure with a polypeptide, a highly biocompatible surface can be obtained. By cross-linking, a culture bed having a stable surface can be obtained throughout the culture period.

By the above method, the surface area to volume ratio is 10 to 300 cm ² / c
A culture bed with m ³ polypeptide surface is obtained. By further applying an animal cell growth promoter on the surface of the polypeptide, cell attachment and growth cost can be increased. As such an animal cell growth promoting agent, fibronectin, a growth promoting factor which specifically works depending on the type of animal cell, a mixture thereof and the like are used. The culture bed after culturing can be regenerated by subjecting it to an alkali treatment such as caustic soda to peel off the polypeptide surface and then forming a new polypeptide surface.

The culture tank used in the present invention is one in which the above-mentioned culture bed is filled in a hermetically-sealed container and is fixed in the container by using a supporting member. As the culture tank, cylindrical vertical type, cylindrical horizontal type,
Although a rectangular type is used, it is usually preferable to use a cylindrical vertical type in terms of easy circulation of the liquid medium. The inner surface of the culture tank is generally a metal surface, especially a stainless steel surface, but other materials may be used. The capacity of the culture tank is appropriately determined according to the final cell number of the target animal cells and the culture characteristics. In the case of a cylindrical vertical culture tank, the ratio of height to inner diameter is usually 0.1-5.
It is better to set the range.

Next, the drawings of the present invention will be described. FIG. 1 shows one embodiment of a culture apparatus system for carrying out the method of the present invention, and FIG. 2 shows another embodiment.

In FIG. 1, reference numeral 1 denotes a cylindrical vertical-type culture tank, in which a culture bed 2 is arranged and fixed by appropriate supporting members 3, 3. A liquid distributor 4 is arranged above the culture bed 2. In this case, the liquid distributor and the supporting member may not be separate, but may be used in common due to their shapes. A constant temperature jacket (not shown) is usually arranged on the peripheral wall of the culture tank. The culture temperature is maintained by controlling the temperature of the liquid culture medium that is circulated with the constant temperature jacket. The culture bed, in order to perform efficient culture,
It is preferable to arrange the cells in the space of the culture tank so as to occupy 50% or more, preferably 90% or more of the volume of the culture tank. The liquid distributor 4 is for uniformly flowing down the liquid medium from the upper part of the culture bed, and is composed of a perforated plate or a tubular body having small holes, and the material thereof can be metal or plastic. The support members 3, 3 are for supporting and fixing the culture bed 2 in the culture tank, and usually, a perforated plate or a net-like member is used, and they are installed at least above and below the culture tank. The shape and size of this support member may be appropriately designed in consideration of strength within the range where the culture bed can be fixed. Also,
Metal or plastic can be used as the material.

The culture bed arranged in the culture tank can be composed of one or more non-woven fabrics or non-woven fabrics cut into small pieces. In the case of comprising multiple non-woven fabrics, in order to prevent compressive deformation of the culture bed. , An appropriate number of support members can be placed between the culture bed stacks at appropriate intervals.

To culture animal cells using the culture apparatus system shown in FIG. 1, first, the animal cells to be grown are seeded on the culture bed. After seeding the animal cells, drive the pump 7 to
The liquid medium flowing through the culture bed 2 is circulated through the line 10 to the culture tank through the medium receiving tank 6, the oxygen supplier 8 and the line 13. By continuously and repeatedly performing such an operation, animal cells are grown on the surface of the polypeptide of the culture bed 2. In this case, a new liquid medium can be supplied in the circulation system, or waste products of animal cells or products for secretion can be partially extracted and processed. The circulation direction of the culture solution may be either vertical or horizontal, and the direction may be appropriately changed during the culture period.

When animal cell culture is performed, supplemental oxygen (or air) is required, but in the present invention, the conventional supplemental oxygen method is (1) through a porous tube (sparger) into a culture solution in a culture tank. To directly blow air, (2) to dissolve oxygen into the culture medium in the culture tank through a silicon tube or a silicon film, (3) to pass air to the surface of the culture medium in the culture tank, and oxygen from the liquid surface , (4) In the liquid medium circulation step, oxygen is supplied through the membrane to the supply medium outside the culture tank, (5) Fluorocarbon in which oxygen is dissolved is directly supplied to the culture solution in the culture tank. However, any method such as a method of bringing the culture solution into liquid / liquid contact to transfer oxygen may be used.
In large-scale culturing of animal cells, it is important that animal cells are sensitive to shearing force and oxygen is effectively supplied without generating shearing force.
(5) is effective.

Fluorocarbon has been studied as an artificial blood in the medical field and has been confirmed to be physiologically harmless to animals. This one has high oxygen solubility,
It is inert to the medium, physiologically harmless to animal cells, has a large specific gravity, and is easily separated from the medium. In the present invention, it can be preferably used as an oxygen supply medium (oxygen carrier). The advantage of using fluorocarbon as an oxygen supply medium is that oxygen (air) is blown into the fluorocarbon liquid outside the culture tank, and the oxygen transfer to the culture medium is due to liquid / liquid contact between the oxygen-saturated fluorocarbon liquid and the liquid medium, which causes foaming. Does not occur, and the shearing force on the cells can be suppressed to a low level. Fluorocarbon has an oxygen solubility of 10 times or more that of water, and thus has an advantage that oxygen can be supplied more effectively than directly blowing oxygen into the liquid medium.

Next, FIG. 2 shows an example in which fluorocarbon is used to supply oxygen. In the reference numerals shown in FIG. 2, the same portions as those shown in FIG. 1 have the same meaning.

In the culture apparatus system shown in FIG. 2, the liquid medium containing the fluorocarbon extracted from the culture tank 1 through the line 10 is introduced into the liquid medium / fluorocarbon liquid separator 20, where the liquid medium and the fluorocarbon are separated. And the liquid medium enters the medium receiving tank 6, from which the pump 7, line
It is introduced into the culture tank 1 through 13 and the valve 28. on the other hand,
The separated fluorocarbon enters the fluorocarbon oxygen enricher 21 through line 23. In this oxygen enricher, sterile air is introduced and dispersed in the fluorocarbon, and oxygen in the air is dissolved in the fluorocarbon. The air that has passed through the fluorocarbon is discharged through the line 24 and the filter 25. Fluorocarbon containing oxygen is pump 22, line
It is introduced into the culture tank 1 through the valve 26 and the valve 27, where it is brought into contact with the culture medium, and dissolved oxygen in the fluorocarbon is transferred into the culture medium. By continuously performing such an operation, animal cells steadily grow on the culture bed 2 without lack of oxygen.

In the device of the present invention, various modifications are possible, for example, instead of the fluorocarbon, another oxygen carrier having an affinity for oxygen can be used, and as a culture tank, a so-called multistage It is possible to use a tray type in which a plurality of plate-shaped culture beds are placed on each tray and the medium perfusate is sequentially flowed down horizontally from the upper tray. Further, the liquid medium can be circulated from below in FIG. Further, in FIG. 2, the liquid medium is circulated from below and the fluorocarbon is circulated from above,
You may make a countercurrent contact.

〔Example〕

 Next, the present invention will be described in detail with reference to examples.

Example 1 The non-woven fabric shown in Table 1 which was washed with acid and alkali and then washed with water was immersed in a 0.2% pepsin-solubilized collagen aqueous solution adjusted to pH 7 with disodium phosphate at 20 ° C for 1 hour to form a collagen surface on the non-woven fabric. It was then air dried.

Then 0.5% with the addition of 20 mmol / l disodium phosphate
The collagen surface was crosslinked and insolubilized by immersing it in an aqueous solution of glutaraldehyde for 10 minutes, then washed with water and dried.

In the culture bed obtained by the above operation, the surface of the constituent fibers is uniformly coated with collagen, and the fact that it has a collagen coating everywhere in the space formed by the fibers shows that it is an optical microscope, a scanning electron microscope. Confirmed in.

Example 2 A glass column having a diameter of 2.7 cm and a length of 50 cm was loaded with 100 layers of the non-woven fabric shown in Table 1 cut into a disc having a diameter of 2.7 cm in a layer form and adjusted to pH 5 with caustic soda to solubilize 0.05% pepsin. 500m collagen aqueous solution at 20 ℃ at a flow rate of 200m / min
It was circulated for a period of time to form a collagen surface on the nonwoven fabric, and then dried with air.

Next, 300 m of 0.2% hexamethylene diisocyanate / methanol solution was circulated for 10 minutes at a flow rate of 200 m / min to crosslink and insolubilize the collagen surface, and then washed with water.

Through the above operation, a large number of culture beds could be manufactured at the same time. In the obtained culture bed, the surfaces of the constituent fibers were uniformly coated with collagen, and the collagen coating was provided everywhere in the space formed by the fibers.

Example 3 The surface of the non-woven fabric shown in Example 1 was washed, treated with caustic soda, washed, neutralized, then dried and cooled to 17 to 20 ° C. On the other hand, a gelatin solution having the following composition was prepared and the temperature was set to 35 to 37 ° C.

Gelatin: 12 parts by weight Glycerin: 40 parts by weight Ethyl alcohol: 150 parts by weight Water: 1000 parts by weight The cooled non-woven fabric is completely immersed in the above solution for 1.5 to 2 minutes, then pulled up, shaken off the gelatin solution sufficiently, and then at 25 ° C.
The membrane was solidified in 3 to 4 minutes by holding it, and then thoroughly dried.

Next, this non-woven fabric coated with gelatin was subjected to a crosslink insolubilization treatment in the same manner as in Example 1.

By the above operation, a culture bed composed of a nonwoven fabric having a crosslinked and insolubilized gelatin surface could be manufactured.

Example 4 The non-woven fabrics shown in Table 1 which were washed with acid and alkali and then washed with water obtained in Example 1 were each cut into 4 mm squares, and 80 pieces of each were cut into glass of 3.7 cm in diameter and cm in length. The sample was placed in a column made of autoclave, sterilized by an autoclave, and then conditioned by flowing 100 m of Ham-F12 medium containing 5% of beef tallow serum.

Seed solution containing 2 x 10 ⁶ Chinese hamster ovary CHO-K1 cells cultured in a flask for 4 days in this column 50m
The seeds were circulated at a flow rate of 2.5 m / min for 1 hour and seeded at 2.1 × 10 ⁵ cells / cm ^{3 for} the culture bed and 2.3 × 10 ⁴ cells / cm ^{3 for the} non-woven fabric.
Cells were attached.

Then, 500m of Ham-F12 medium containing 5% of beef tallow serum was added to 2.5m.
When circulation culture was carried out at 37 ° C while circulating at a flow rate of / min, 1.2 × 10 ⁷ cells / cm ³ in the culture bed after 5 days of culture,
In the non-woven fabric, 7.4 × 10 ⁵ cells / cm ³ of cells were growing. In this case, the culturing operation was performed in a CO ₂ incubator, a part of the circulation pipe was a silicon tube, and oxygen was supplied through the silicon tube.

As a result, the efficiency of the culture bed made of the nonwoven fabric having a collagen surface was found to be about 10 times as high as the adherence rate of seeded cells and about twice as fast as the cell growth rate.

Example 5 Nine non-woven fabrics obtained in Example 2 were treated with a diameter of 2.7 cm and a length of 4 cm.
The glass column was loaded in layers and sterilized by an autoclave, and 100 m of Ham-F12 medium containing 5% beef tallow serum was flowed and conditioned.

Seeding solution containing 1 x 10 ⁶ Chinese hamster ovary CHO-K1 cells pre-cultured in a flask for 4 days in this column 5
After seeding by circulating 0 m at a flow rate of 2.5 m / min for 1 hour,
500 m of Ham-F12 medium containing 5% of beef tallow serum was circulated at a flow rate of 2.5 m / min while culturing at 37 ° C. for 4 days while supplying oxygen through a silicon tube in a CO ₂ incubator as in Example 4. Then, the medium was exchanged, and the cells were further cultured for 2 days. After the culture was completed, a 0.25% trypsin solution was flown through the column to collect the grown cells, and 3.3 × 10 ⁸ cells were obtained.

The cells were grown about 300 times by the above culture operation, and the cell density at that time was about 1.6 × 10 ⁷ cells / cm ³ of the culture bed.

〔effect〕

According to the present invention, it is possible to cultivate a large amount of animal cells at high density, which has been conventionally difficult to perform high-density culturing and mass culturing. In particular, in the present invention, a specific non-woven fabric having a polypeptide surface is used as the culture bed, which is arranged and fixed in the culture tank, which enables efficient animal cell culture, and the structure of the culture tank. It has the advantage of being easy to design and greatly simplifying the exchange of culture medium, which is frequently performed in animal cell culture. Furthermore, when oxygen is supplied to the medium by using an oxygen carrier such as fluorocarbon, the effect of shearing force on the cells, which is not preferable for animal cell culture, and the foaming of the medium can be avoided. It can be done smoothly and objectively.

[Brief description of drawings]

FIG. 1 shows an animal cell culture device system, and FIG. 2 shows a modified example thereof. 1 ... Culture tank, 2 ... Culture bed, 3 ... Culture bed support member,
4 ... Liquid distributor, 6 ... Culture liquid receiving tank, 8 ... Oxygen supply device, 20 ... Culture liquid / fluorocarbon liquid separator, 21 ... Fluorocarbon oxygen enricher.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Keiichi Anraku 3-1-8, 3102-242 Takeyama, Midori-ku, Yokohama-shi, Kanagawa Prefecture (56) References JP-A-61-271987 (JP, A) JP-A-57 -501411 (JP, A) JP 60-13145 (JP, B2)

Claims (4)

[Claims] 1. In a method of culturing animal cells under oxygen supply, a nonwoven fabric having a porosity of 90 to 98% and having a polypeptide surface and a thickness of 0.03 to 0.3 mm is used as a culture bed, and a liquid medium is used. A method for culturing animal cells, which comprises culturing while circulating in the culture bed, and circulating the liquid medium circulating in the culture bed to the culture bed. 2. The method according to claim 1, wherein the polypeptide is collagen or gelatin. 3. A thickness of 0. with a polypeptide surface inside.
Non-woven fabric with a porosity of 90-98% consisting of 03-0.3 mm fibers,
An animal cell culture vessel comprising a support member for supporting and fixing the nonwoven fabric. 4. The culture tank according to claim 3, wherein the polypeptide is collagen or gelatin.