WO2003085080A1

WO2003085080A1 - Cell culture plate and system using the same

Info

Publication number: WO2003085080A1
Application number: PCT/KR2002/000603
Authority: WO
Inventors: Kwang-Hyun Yoo; Sung-Ho Shin
Original assignee: COREBIOTECH Co Ltd
Current assignee: COREBIOTECH Co Ltd
Priority date: 2002-04-04
Filing date: 2002-04-04
Publication date: 2003-10-16
Anticipated expiration: 2004-10-04
Also published as: AU2002246422A1

Abstract

The invention concerns a cell culture plate comprising: (a) a bottom plate with a central air hole for allowing flow of air; (b) a sidewall formed around the bottom plate; (c) a protuberant air tube mounted around the central air hole; and (d) a culture medium supply tube mounted in a portion of the bottom plate adjacent to the sidewall, the culture medium supply tube being designed so that when joining a plurality of the cell culture plates one above another, the lower part of the culture medium supply tube of the upper cell culture plate, and cell culture system comprising the cell culture plates joined together one above another, and method using them.

Description

CELL CULTURE PLATE AND SYSTEM USING THE SAME

TECHNICAL FIELD

The present invention relates to a cell culture plate for use in culturing of animal cells and a system using it, and more particularly a plate, system and method for culturing animal cells to provide an improved culturing time and productivity.

BACKGROUND ART

"Cell Factory" (manufactured by The NUNC Company) well known as a system for culturing animal cells comprises a plurality of rectangular cell culture plates laid one above another, which are uniformly supplied with cells and culture media to perform static culture, control the culture environment by diffusing air through the air tube and culture medium supply tube mounted on the upper part to communicate with the cell culture plates, and exchange the culture medium in batch. Cell Factory has many advantages in performing static culture for the initial growth of cells, but hardly control the culture environment for the prolonged culturing period subsequent to the initial growth of cells as well as the amount of lactic acid much produced during the static culture. Also well known in this art is the roller bottle for another system for culturing animal cells. This is a bottle- shaped vessel supplied with a culture medium and cells, which is continuously rotated to cause the cells to be attached to the inside thereof for growing. The rotation of the roller bottle causes the cell surface to make rotational movements between the inside and outside of the culture medium, so that the cells repeat the process of absorbing nutrients in the inside and oxygen in the outside. This process results in the exhaustion effect to enable effective use of carbon sources as well as reduction of the amount of lactic acid produced. However, the number of the roller bottles must be increased with the scale of cell culturing, thus making it difficult to control the culture environment.

DETAILED DESCRIPTION OF THE INVENTION

Endeavoring to resolve the problems of such conventional cell culturing, the inventors have developed a cell culture plate and system that may easily control the culture environment together with having all the advantages of the cell culture using the flask and roller bottle.

Accordingly, it is an object of the present invention to provide a cell culture plate for culturing animal cells. It is another object of the present invention to provide a system for culturing animal cells comprising a plurality of the present culture plates joined one above another, which may effectively control the culture environment .

It is still another object of the present invention to provide a method of culturing animal cells.

According to one aspect of the present invention, there is provided a cell culture plate, which comprises: (a) a bottom plate with a central air hole for allowing flow of air; (b) a sidewall formed around the bottom plate; (c) a protuberant air tube mounted around the central air hole; and (d) a culture medium supply tube mounted in a portion of the bottom plate adjacent to the sidewall, the culture medium supply tube being designed so that when joining a plurality of the cell culture plates one above another, the lower part of the culture medium supply tube of the upper cell culture plate communicates with the inside of the lower cell culture plate.

Fig. 1 is a perspective view for illustrating a system for culturing cells according to an embodiment of the present invention, and Figs. 2A to 2C are respectively perspective, cross sectional and plane views for illustrating a cell culture plate used for constructing the cell culture system of Fig. 1. As shown in the drawings, the cell culture system 1 according to an embodiment of the present invention is obtained by joining a plurality of cell culture plates 10 one above another.

A cell culture plate 10, as shown in Figs. 2A to 2C, comprises a circular bottom plate 12, sidewall 14 formed around the bottom plate 12, air tube 16 mounted at the center of the bottom plate 12, and culture medium supply tube 18 mounted at a portion of the sidewall 14.

The bottom plate 12 is provided with a circular central air hole 13 for allowing flow of air. The bottom plate 12 may be made wholly flat, or sloping from the air hole 13 towards the sidewall 14 or radially. The bottom plate may have the upper surface provided with a plurality of creases (not shown) , or coated with a fibrous or sponge material (not shown) . This serves to increase the size of the upper surface of the bottom plate 12, thus facilitating the attachment of the cells thereto as well as improving the property to hold the cells.

A material suitably used for coating the bottom plate of the present cell culture plate may be poly-L-lysine, plasma, etc. to facilitate the attachment of the cells. According to an embodiment of the present invention, the bottom plate may be made to have a circular, elliptical or five or more sided polygonal shape, and most preferably a circular shape. Such a shape of the bottom plate serves to prevent eddying and shearing flows of the culture medium that exert stresses to the cells in the culture medium impeding the growth of the cells.

The air tube 16 protrudes upwards from around the air hole 13 of the bottom plate 12, preferably having a truncated conic shape with the top end diameter smaller than the base diameter.

The culture medium supply tube 18 is mounted in a portion of the bottom plate 12 adjacent to the sidewall 14. Further, the culture medium supply tube 18 is designed so that when joining a plurality of the cell culture plates 10 one above another, the lower part of the culture medium supply tube 18 of the upper cell culture plate communicates with the inside of the lower cell culture plate. Preferably, as shown in the drawings, the culture medium supply tube 18 has the upper portion slanted approaching the outside of the cell culture plate 10. Although the culture medium supply tube 18 is arranged in the inside of the sidewall in Figs. 2A to 2C, it may be also arranged in or outside the sidewall.

The culture medium vessel 10 is made of a glass or plastic, and preferably a transparent material. This facilitates observation of the growth of the cells cultured. In the present embodiment, the cell culture plate 10 has been described as having circular shape, and the air tube 16 and culture medium supply tube 18 as having truncated conic shape. However, these are not limited to such shapes, but may have elliptical and truncated elliptical conic shapes, or polygonal and truncated polygonal pyramidal shapes .

The cell culture plate may have various sizes, and typically a diameter of 5 to lOOCm and a height of 0.3 to 5Cm. Preferably, the height of the culture medium supply tube 18 is higher than that of the sidewall 14. This serves to prevent the culture medium in the cell culture plate 10 from reversely flowing into the culture medium supply tube 18.

According to another aspect of the present invention, there is provided an animal cell culture system comprising a plurality of the present cell culture plates joined one above another.

According to a preferred embodiment of the present invention, the system for culturing cells comprises: (a) a plurality of the cell culture plates joined one above another with each internal space sealed, each cell culture plate having a bottom plate and a sidewall formed around the bottom plate; (b) an air passage formed through the centers of the joined cell culture plates so as to communicate with the internal space of each of the cell culture plates with air inlet and outlet openings; and (c) a culture medium supply passage formed in parallel with the air passage along the sidewalls of the joined cell culture plates so as to communicate with the internal space of each of the cell culture plates with culture medium inlet and outlet openings.

The present cell culture system resolves the disadvantages of the conventional static culture and roller bottle culture without sacrificing their advantages.

Describing the present cell culture system, the parts or elements in common with the cell culture plate are omitted from description in order to avoid complication. The cell culture plates 10 are joined one above another to construct a system for culturing cells according to an embodiment of the present invention, as shown in Fig. 1. The joining between the adjacent cell culture plates is accomplished by uniting the bottom plate 12 of the upper cell culture plate 10 and the sidewall 14 of the lower cell culture plate 10 by means of an adhesive or thermally melting.

The air tubes 16 of the cell culture plates 10 are aligned to form the air passage 20, and the culture medium supply tubes 18 to form the culture medium supply passage 30.

According to a preferred embodiment of the present invention, the upper and lower ends of the air passage 20 are provided with a gas filter 21. The gas filter serves to filter germs contained in an air mixture. Preferably, the upper end of the air passage 20 is connected with an air hose (not shown) connected to an air pump. These air supply devices supply C0₂ gas suitable for the present cell culture system, which serves to positively control the hydrogen ion concentration of the culture medium in the system.

Preferably, there is provided a connecting tube to facilitate connection between the air hose and air passage 20 and to minimize air leakage.

Preferably, the air passage 20 is provided with an auxiliary flow tube crossing with the air tube of the lower one of the adjacent cell culture plates so as to facilitate diffusion of air into the cell culture plate.

Preferably, the culture medium supply passage 30 has a lower end connected to a culture medium inlet tube 31 to supply the culture medium, and an upper end connected to a culture medium outlet tube 32 to withdraw the culture medium. It is preferable to provide the ends of the culture medium supply passage with devices for preventing reverse flow of the culture medium.

Preferably, the bottom plates 12 of the cell culture plates 10 are provided with at least one protuberance or protruding wall so as to prevent bending of the bottom plates 12 of the cell culture plates 10 joined one above another and to maintain the interval between two adjacent ones .

Preferably, the cell culture system further includes at least one of sloping means for sloping the system, directional change means for changing the direction of the slope of the system, rotational means for rotating the system, and vertical and horizontal change means for changing the position of the system between vertical and horizontal positions. These means enables the present cell culture system to be simply automatized.

The present cell culture system is operated in the following way to culture animal cells. Firstly, the cell culture system 1 is laid into horizontal position with the culture medium supply passage 30 being at the lowest position, as shown in Fig. 3, into which the culture medium is supplied through the culture medium inlet tube 31. In this case, the culture medium outlet tube 32 is blocked off. Accordingly, the culture medium is supplied through the culture medium supply passage 30 into the internal spaces of the cell culture plates 10.

When the internal space of the cell culture plate 10 has been supplied with the culture medium to a prescribed level, the cell culture system 1 is turned into vertical position to spread the culture medium uniformly over the internal space of each cell culture plate 10. In this state, the static culture is performed to induce attachment of the cells to the internal surface of the cell culture plate and their initial growth (Fig. 4) . Meanwhile, the C0₂ concentration of the air supplied by the air pump is controlled to a prescribed level (typically 0-10% C0₂) to control the culture environment of the internal space of the cell culture plate 10.

Completing the static culture to obtain the initial growth of the animal cells on the culture surface, the sloping culture is performed. As shown in Fig. 5, the sloping culture is performed by slowly rotating the cell culture system 1 sloped at a given angle or by continuously changing its slope so that the cells are alternately immersed into and deprived of the culture medium.

In this case, the rotation of the culture medium is made at a speed of about 1 to 1/8 rpm, more preferably about 1/2 to 1/3 rpm. In order to enable the culture medium to uniformly contact the culture surface in the cell culture plate during the sloping culture, the slope of the cell culture system is controlled for the culture medium to contact the bases of the air tubes of the air passage 20 and to cover about 1/3 to 2/3 parts of the bottom plate of the cell culture plate.

While the sloping culture results in the exhaustion effect to prevent the culture medium from increasing hydrogen ion concentration, it is desirable to continuously or intermittently supply C0₂-containing air during this process .

Exchange of the culture medium is carried out by the steps of laying the cell culture system 1 into horizontal position with the culture medium supply passage 30 being at the lowest position, as in the initial cell culturing, withdrawing the used culture medium through the culture medium outlet tube 32, supplying fresh culture medium through the culture medium inlet tube 31, rotating the cell culture system by 180°, and slowly erecting it into vertical position.

The instant cell culture system may easily control the culture environment, having all the advantages of both the static culture using the flask and the culture using the roller bottle.

The instant cell culture system enables the cell culture surface to be intermittently supplied with nutrients, thereby resulting in effective use of the nutrients by the exhaustion effect as well as restraining the culture medium from significantly increasing hydrogen ion concentration. In addition, suitable amount of C0₂ is supplied through the air passage to control the culture environment in the cell culture system, thus continuously maintaining the culture environment adapted to the cell culture. Furthermore, the air is smoothly diffused through the air passage not so as to generate air bubble stresses like a bioreactor.

The present cell culture system includes a plurality of cell culture plates laid one above another to enable large-scale culture, facilitates exchange of culture medium, and may fully automatize the cell culture by means of an automatic system operable under sterilized condition. Especially, the present cell culture system is suitable for subsequent long-term culture because of the culture environment easily controlled by flow and diffusion of air in the cell culture plates.

Besides, the present cell culture system provides a cell culture period much prolonged compared to a conventional one. Moreover, it maximizes the efficiency of using the nutrients in the culture medium, significantly improving the efficiency of culturing animal cells.

In another aspect of the present invention, a method of culturing animal cells, comprises the steps of: (a) inoculating said animal cells in a cell culture plate; (b) performing static culture by setting the cell culture plate horizontal to induce the attachment of the animal cells to the surface of the cell culture plate and their growth; (c) sloping the cell culture plate containing the culture medium having completed the static culture so as to cause a part of the bottom plate of the cell culture plate to be covered by the culture medium and the remaining part to be exposed directly to air; and (d) performing sloping culture by continuously changing the sloping direction of the cell culture plate or rotating it to induce rotation of the culture medium.

Hereinafter will be described the present cell culture method with omitting descriptions common to the cell culture plate and cell culture system for convenience's sake.

The present method overcomes the disadvantages inherent in both the conventional static culture and roller bottle culture without sacrificing their advantages. Namely, it guarantees the property of the static culture contributing to the initial cell growth and the property of the roller bottle culture contributing to the final production of cells by the exhaustion effect.

According to the method, firstly are inoculated animal cells of interest in a cell culture plate. Any kind of culture mediums usually used in the art may be used, and a suitable kind may be easily determined according to various factors such as the kind of a target animal cell .

The present method may culture any kind of animal cells, preferably animal cells attachable to the surface of the cell culture plate.

After inoculating is performed the static culture to induce the attachment of the animal cells to the surface of the cell culture plate and their initial growth. This static culture is almost the same as that using the conventional cell culture flask.

After the initial growth of animal cells is performed the sloping culture.

The sloping culture causes the cell surface on the culture medium surface to directly contact the air to effectively receive oxygen, thus facilitating sugar metabolism. Lacking in oxygen, glucose is degraded into lactic acid for generation of energy for the cells, thus increasing the hydrogen ion concentration in the culture medium. The increased hydrogen ion concentration is liable to damage cells. However, sufficient amount of oxygen supplied causes the glucose to enter into TCA cycles, generating much energy for cells as well as preventing increase of the hydrogen ion concentration.

Since the glucose as energy source (carbon source) in the culture medium rapidly exhausted in the cell surface directly contacting the air, the cells themselves cannot use but TCA cycles for effective use of the energy source (carbon source) . However, this situation prolonged causes the cells themselves to be subjected to stresses, but this problem is resolved by returning into the culture medium of the cells.

As described above, the sloping culture causes the cells to move alternately to the inside and outside of the culture medium, so that the cells absorb sufficient amount of the nutrients in the inside, and sufficient amount of oxygen in the outside to generate the exhaustion effect enabling the effective use of the nutrients, especially the carbon source (energy source) , resulting in the supply of sufficient energy source and organic molecules. In addition, the exhaustion effect effectively eliminates the lactic acid from the culture medium, or inhibiting its production. According to a preferred embodiment of the present invention, although the sloping culture causes the exhaustion effect to prevent increase of the hydrogen ion concentration in the culture medium, it is desirable to continuously or intermittently inject C0₂-containing air during the culture. The C0₂ concentration in the C0₂- containing air may be varied according to the hydrogen ion concentration in the culture medium, typically 0 to 10%. The C0₂-containing air positively may control the culture environment to establish the optimum culture condition.

The instant method significantly prolongs the period to culture, maximizes the efficiency of the nutrients in the culture medium, and significantly improves the efficiency of the animal cell culture.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a perspective view for illustrating a system for culturing cells according to an embodiment of the present invention;

Figs. 2A to 2C are respectively a perspective, cross sectional, and plane views for illustrating a cell culture plate used in constructing the cell culture system of Fig.

1;

Fig. 3 is a cross sectional view of the present cell culture system laid horizontal to feed culture medium;

Fig. 4 is a cross sectional view of the present cell culture system erected vertical for the static culture;

Fig. 5 is a cross sectional view of the present cell culture system sloped for the sloping culture;

Fig. 6 is a graph for illustrating the characteristics of CHO cells cultured by the present cell culture system; and

Fig. 7 is a graph for illustrating the characteristics of C127 cells cultured by the present cell culture system.

EXAMPLES

The following specific examples are intended to be illustrative of the invention and should not be construed as limiting the scope of the invention as defined by appended claims .

Comparative Example 1: Culture Properties of CHO Cells Based on Culture System

In order to evaluate the performance and culture properties of the present culture system, a determination was made of cell morphology, lactate production rate to consumed glucose amount, available culture time and culture yield using gene-manipulated CHO cells in conventional flask, roller bottle and bioreactor. The CHO cells are currently used as host for production of drug in the art and in this example they are transformed for production certain cytokine , The used media is DMEM/F12 (1 : 1) containing 10% FBS (Gibco Cat No. 12400) . The results are given in Table 1.

Table 1 Culture Properties of Transformed CHO Cells According to

Culture System

Cell morphology of cells cultured was observed in inverted microscope. Lactate production rate relative to consumed glucose was measured with Glucose kit (Sigma Cat . No. 510-A) and Lactate kit (Sigma Cat. No. 826-A) . Productivity of cytokine was measured according to ELISA method (Langone, J. J. et al . , Immunochemical Techniques, Part A. Methods in Enzymology, 92, Academic Press).

Comparative Example 2: Culture Properties of C127 Cells

Based on Culture System

In order to evaluate the performance and culture properties of the present culture system, cell morphology, growth location, lactate production rate to consumed glucose amount, productivity and available culture time were observed and measured using transformed C127 cells in conventional flask, roller bottle, bioreactor and hollow fiber. The results are given in Table 2.

Table 2 Culture Properties of Transformed C127 Cells According to

Culture System

As seen in Tables 1 and 2, the productivity of cells cultured varies depending on the principle of culturing (e.g., method for sugar degradation, atmosphere control method and cell morphology) . It is believed that the increased productivity and extended culture time of the roller bottle is attributed to the fact that oxygen exchange is actively occurred between cell surface and air within the roller bottle, temporary exhaustion of glucose as carbon source allows the cells to effectively use the carbon source, and cells are transferred into the medium to absorb a sufficient amount of nutrients so that cell activity is continuously maintained.

In the flask, cells are grown in the medium and subjected to static culture, adhering to the bottom. This culture system is efficient so long as cells grow at a low density. However, it is not easy for oxygen to penetrate into the medium contained in a flask. Further, the cycle from supernutrition to innutrition (that is, medium exchange cycle) is so long that the cells are liable to be damaged by high cell density, in addition to having difficulty in efficiently using carbon source.

On the other hand, when culturing is carried out with microcarriers serving as a matrix or cells being suspended, dissolved oxygen levels are increased. In this case, however, the cells undergo changes in cell morphology so that carbon sources cannot be efficiently utilized, reducing the productivity. Particularly, the lactate thus produced acidifies the medium, making the medium exchange cycle shorter. Further, desired biomaterials produced as a result of gene manipulation are generally vulnerable to low pH, which results in reducing the productivity.

EXAMPLE 1: Cell Culture Using the Cell Culture System of the

Present Invention

In order to examine the performance principle and capacity of the present cell culture system comprising a plurality of circular cell culture plates, Pyrex glass and Teflon tubes are combines so as to form a cell culture system with a shape and structure as shown in Fig. 1.

The inventors constructed various kinds of circular cell culture plates with different overall shapes, e.g., sloped funnel-shaped, and flat as shown in Fig. 1. The test proved the circular flat-bottomed cell culture plate to be the most suitable in order to continuously perform both the static and the sloping culture in single cell culture system.

The cell culture system for this test was constructed by employing five cell culture plates with a diameter of 25 cm. The bottom of each cell culture plate was coated with 2% gelatin solution (Sigma G1393) over 2 hours so as to facilitate the attachment of the cells. Then, .the five culture plates were joined together by applying non-toxic silicon bond to their junctions to form a single unit, cleaned and sterilized.

The cells used in the culture were CHO cells (ATCC CCL-61) mostly used in production of biomedicines and C127 cells (ATCC CRL-1804) rich in the characteristics of the fibroblast of adhesive cells. The culture medium for CHO cells was obtained by adding 2.438 g/L of NaHC0₃ to the DMEM/F12 (1:1) (Gibco, Cat. No. 12400) and then adjusting pH to 7.2, which was then sterilized and filtered, maintained at 4°C. The culture medium for C127 cells was obtained by adding 3.7 g/L of NaHC0₃ to the DMEM high glucose and then adjusting pH to 7.2, which was then sterilized and filtered, maintained at 4°C. The fetal bovine serum (FBS Gibco, Cat. No. 26140-079) was added to the medium to be 10% (v/v) .

The cell culture system constructed was laid horizontal, and fed with the culture medium with an initial cell concentration of 7.5 x 10^δ/lOO ml through the culture medium inlet tube 31. The cell culture plate only for the static culture was supplied with 100 ml of the culture medium while the cell culture system comprising 5 cell culture plates was supplied with 400 ml of the culture medium. Thereafter, the cell culture system was rotated by 180°C so as to position the culture medium supply passage uppermost, and then, slowly erected vertical.

After inoculating, the cells were cultured for two days at 37°C under 5% C0₂ for the attachment and growth, and then, exchanging the medium with fresh medium, subjected to the static culture for three days to fully cover bottom of cell plate. Thereafter, while continuously subjecting only to the static culture for the control, the experimental set according to the present invention was subjected to the sloping culture, with the culture medium being exchanged every two days. For the sloping culture, there was provided an auxiliary device for slowly rotating the culture medium counterclockwise by changing the sloping direction. The sloping culture was performed with the circulation speed of the culture medium at 0.4 rpm. The culture medium was sampled to investigate the culture characteristics (metabolism, cell shape, etc.).

The results proved that the present cell culture system contributed to better growth pattern of animal cells and improved productivity. The present cell culture system may have a size suitable for convenient performance in the following manner. Geometrically considering the height of the culture medium in the sloping culture of the present culture system comprising a plurality of the circular cell culture plates, when a given part of the cell culture plate is covered with a constant amount (v) of the culture medium per unit culture surface, the height h of the culture medium at the end toward the sloping direction is always the same (h=kv; k=constant) . Namely, if the amount of the culture medium per unit culture surface is the same regardless of the diameter of the cell culture plate, the height h is also the same for all sizes of the diameter with the sloping angle only changed. Thus, it is possible to increase the diameter of the cell culture plate with the height maintained constant .

The instant cell culture system enabled the culture medium to be quickly exchanged through the culture medium inlet and outlet tubes, and provided smooth air circulation therein. In addition, while the circulation speed of the culture medium was increased to 2 rpm in order to evaluate the possibility of the cells physically damaged by rotation of the culture medium on the culture surface during the sloping culture, there was not detected any change of the cell's property. This test proved that the cells might be cultured by the cell culture system comprising circular cell culture plates even with a diameter of 50 cm.

EXAMPLE 2 : Characterization of Culture Using the Cell Culture System of the Present Invention

According to the method as described in Example 1, the cells were inoculated into the present cell culture system, and then cultured for 31 days, in order to investigate the culture characteristics such as cell metabolites. The amount of the glucose and lactic acid and the cell shape were measured and observed using the same method as described in the comparative experimental example 1.

The initial cell attachment and growth was well accomplished by the static culture, and the prolonged culture was carried out by the sloping culture, generating the culture characteristics significantly different from those of the static culture. In addition, the culture characteristics varied depending on the cells cultured. The sloping culture showed high efficiency in using the carbon source .

As shown in Fig. 6, the CHO cell culture resulted in higher increase of the entire amount of metabolism in the sloping culture than in the static culture. Further, the present sloping culture increased the amount of glucose absorbed but decreased the amount of lactic acid generated, efficiently using the carbon source, compared to the static culture. Moreover, indirectly measuring the cell concentration by staining the cell surfaces with crystal violet-formalin, significant increase of the cell concentration was observed in the present sloping culture. Calculating the amount of glucose absorbed and lactic acid generated in a circular cell culture plate for 31 days, the former was 2.843 g in the static culture and 3.052 g in the present sloping culture, while the latter was 1.979 g in the static culture and 1.306 g in the present sloping culture. Using these values to calculate the ratio of the amount of lactic acid generated to the amount of glucose absorbed (consumed), the static culture gave 69.5%, but the present sloping culture gave 42.8%, proving the efficiency of using the carbon source. In addition, this efficiency enabled the hydrogen ion concentration differences of ■ the culture mediums to be observed.

In culturing C127 cells, as shown in Fig. 7, the static culture had the cell surface peeled on the 16^th day causing the test to be stopped, but the present sloping culture made it possible to continue culturing for 31 days. Staining the cell surface with crystal violet-formalin on the 14^th day, the present sloping culture showed multi- layered cells of fibroblast hardly observable in the static culture as well as considerably high cell concentration compared to the static culture. In the present sloping culture, the production of lactic acid was rapidly reduced from the 7^th day since starting of the culture, resulting in the lactic acid concentration of zero, which clearly proved the exhaustion effect induced by the present culturing method. Especially, though there is residual glucose, the absorption of lactic acid was observed representing the cell characteristics of positively employing the lactic acid for the cell metabolism in a given condition. Such cell culture characteristics made no difference in the hydrogen ion concentration in the culture medium of the present sloping culture both directly after and before exchanging the culture medium, while the static culture continuously increased the lactic acid concentration and thus the hydrogen ion concentration to damage the cells, finally leading to their detachment from the surface of the bottom plate.

Therefore, it is understood that the present cell culture system has the following advantages: (a) inducing effectively the initial attachment and growth of cells through the static culture, and increasing the number of cells induced by the effective cell metabolism through the sloping culture, and thus productivity; (b) enabling circulation of a mixed air so as to positively control the culture environment; and (c) minimizing the interval between adjacent cell culture plates to maximize the culture area per unit space, thus dramatically enhancing the efficiency of large scale cell culture.

Claims

WHAT IS CLAIMED IS:

1. A cell culture plate for culturing animal cells, comprising:

(a) a bottom plate with a central air hole for allowing flow of air;

(b) a sidewall formed around said bottom plate;

(c) a protuberant air tube mounted around said central air hole; and

(d) a culture medium supply tube mounted in a portion of said bottom plate adjacent to said sidewall, said culture medium supply tube being designed so that when joining a plurality of said cell culture plates one above another, the lower part of the culture medium supply tube of the upper cell culture plate communicates with the inside of the lower cell culture plate.

2. The cell culture plate as defined in claim 1, wherein said bottom plate is made to have a circular, elliptical or five or more sided polygonal shape in order to prevent eddying and shearing flows of a culture medium.

3. The cell culture plate as defined in claim 1, wherein said bottom plate is sloped from said central air hole towards said sidewall .

4. The cell culture plate as defined in claim 1, wherein said bottom plate has the upper surface provided with a plurality of creases.

5. The cell culture plate as defined in claim 1, wherein said bottom plate has the upper surface coated with a fibrous or sponge material .

6. The cell culture plate as defined in claim 1, wherein said air tube and culture medium supply tube are made to have truncated circular or elliptical conic or pyramidal shape .

7. The cell culture plate as defined in claim 1, having an average diameter of 5 to 100 cm and a height of 0.3 to 5 cm.

8. The cell culture plate as defined in claim 1, wherein the height of said culture medium supply tube is higher than that of said sidewall.

9. A system for culturing animal cells, constructed by joining one above the other a plurality of the cell culture plates as defined in any one of claims 1 to 8.

10. The system for culturing animal cells as defined in claim 9, comprising:

(a) a plurality of the cell culture plates joined one above another with each internal space sealed, each cell culture plate having a bottom plate and a sidewall formed around said bottom plate;

(b) an air passage formed through the centers of the joined cell culture plates so as to communicate with the internal space of each of said cell culture plates with air inlet and outlet openings; and

(c) a culture medium supply passage formed in parallel with said air passage along the sidewalls of the joined cell culture plates so as to communicate with the internal space of each of said cell culture plates with culture medium inlet and outlet openings.

11. The system for culturing animal cells as defined in claim 10, wherein said air passage comprises a plurality of protuberant air tubes each mounted around a central air hole formed in the bottom plate of each cell culture plate.

12. The system for culturing animal cells as defined in claim 10, wherein said culture medium supply passage comprises a plurality of culture medium supply tubes, each of said culture medium supply tubes having a part positioned on the outside of said sidewall and the remaining part positioned in the inside thereof, and being shaped so that when two of more cell culture plates are joined one above another, the lower part of the upper culture medium supply tube communicates with the internal space of the lower cell culture plate.

13. The system for culturing animal cells as defined in claim 10, wherein the upper end of said air passage is provided with a gas filter.

14. The system for culturing animal cells as defined in claim 10, wherein the air tubes forming said air passage are arranged so as to overlap each other in order to facilitate diffusion of air into the cell culture plates.

15. The system for culturing animal cells as defined in claim 10, wherein said cell culture plates are made of a glass or plastic.

16. The system for culturing animal cells as defined in claim 10, wherein the bottom plates of said cell culture plates are provided with at least one protuberance or protruding wall to maintain the interval between adjacent cell culture plates or prevent bending of the bottom plate.

17. The system for culturing animal cells as defined in claim 10, further including at least one of sloping means for sloping said system, directional change means for changing the direction of the slope of said system, rotational means for rotating said system, and vertical and horizontal change means for changing the position of said system between vertical and horizontal positions.

18. A method of culturing animal cells, comprising the steps of :

(a) inoculating said animal cells in a cell culture plate;

(b) performing static culture by setting said cell culture plate horizontal to induce the attachment of said animal cells to the surface of said cell culture plate and their growth;

(c) sloping said cell culture plate containing the culture medium having completed the static culture so as to cause a part of the bottom plate of said cell culture plate to be covered by said culture medium and the remaining part to be exposed directly to air; and

(d) performing sloping culture by continuously changing the sloping direction of said cell culture plate or rotating it to induce rotation of said culture medium.

19. The method of culturing animal cells as defined in claim 18, wherein said animal cells have a property to attach to the surface of said cell culture plate.

20. The method of culturing animal cells as defined in claim 18, wherein said bottom plate is made to have a circular, elliptical or five or more sided polygonal shape in order to prevent eddying and shearing flows of said culture medium.

21. The method of culturing animal cells as defined in claim 18, wherein said method is accompanied during culture by continuous or intermittent supply of C0₂-containing air.

22. The method of culturing animal cells as defined in claim 18, wherein all of said steps are performed in a single unit .