JPH11243948A - Cell culture bed substrate for proliferation of animal cell and its preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective cell culture bed substrate for the proliferation of animal cells and usable as a substitute for collagen. SOLUTION: A membrane composed of silk fibroin or silk sericin selected according to the kind of the animal cell to be cultured or containing both components at a specific ratio is bonded to the substrate of a cell culture bed. The bonding can be carried out by making the lower layer to be insoluble in water, bonding the upper layer and making the layer to be insoluble in water.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell culture bed substrate for culturing animal cells and a method for preparing the same, and particularly to a cell culture suitable for efficiently attaching and proliferating even floating animal cells to the substrate surface. The present invention relates to a bed and a method for preparing the same.

[0002]

2. Description of the Related Art In recent years, with the advancement of biotechnology, new cell fusion and gene transfer technologies have been developed one after another. In connection with this, technologies for culturing a large amount of animal cells and technologies for culturing animal cells have been developed. Research and development for industrially producing useful bioactive substances such as human therapeutic vaccines, interferons, and cell growth factors by genetic engineering techniques that fuse gene transfer techniques have been actively conducted. That is, if a technology for efficiently expressing an exogenous gene by further genetically manipulating animal cells can be further developed, a new useful substance can be efficiently produced. There is a great social demand to develop a cell culture bed substrate that can efficiently grow on a large scale. In particular, the concept of "animal" or "living body" as used herein includes "insects". If an effective culture bed substrate for insect cells is developed, it will be effective in determining toxic substances. It is thought to demonstrate.

[0003] However, mass cultivation of animal cells requires various contrivances depending on whether they are floating or adherent in a medium. For example, lymphoblast cells, which are blood cell cancer cells, or established cell lines such as carcinoma and sarcoma are floating cells that grow in a floating state, and must be aerated and stirred in a culture solution. In this aeration / agitation culture, it is necessary to control the mixing ratio and partial pressure of CO ₂ , O ₂ , N ₂ , air, and the like, and to control dissolved oxygen, and the apparatus configuration is complicated and expensive. Further, culturing large amounts of planktonic animal cells requires a long period of time, and in this process, fatal problems such as microbial contamination occur. Poor cultivation efficiency has also been confirmed in the inventors' experimental examples in which the final cell density did not reach 10 ⁷ cells / ml even in batch culture operations for performing various controls.

[0004] Large-scale culture of adherent cells is relatively easy for these suspended cells, but most of the adherent cells grow only when they adhere to the surface of the culture vessel. Proliferation was inefficient and tended to be rather costly. Therefore, special-type culture devices such as microcarrier culture method and hollow fiber culture method that take large attachment area have been developed, but from the viewpoint of simplicity, adherent cells can be efficiently cultured in ordinary culture vessels such as flasks. Hopefully you can.

[0005] Next, in order to culture cells efficiently and economically irrespective of their properties such as floatability and adherence, nutrients are replenished to the medium as needed, and the culture solution is set in an environment suitable for cell growth. As a result, the medium had to be changed frequently, which hindered efficient cell culture. In order to continue the cell culture, when the cells are grown to a target amount, it is necessary to separate only the cells or the cells from the useful medium, and an operation using a centrifuge or a decantation method is used. From the standpoint of this cell / medium separation, adherent cells with better adherence to the substrate surface are more convenient than floating cells lacking adherence to the substrate.

[0006]

SUMMARY OF THE INVENTION Based on the above,
Focusing on conventional methods that have been attempted to promote cell growth on cell culture bed substrates, a method of applying a plasma treatment to the substrate surface to give a charge, immobilizing cell growth factors on the substrate surface And a method of thinly coating collagen on the surface of the substrate. However, the plasma processing method requires a large and expensive plasma processing apparatus, and the trouble of isolating and identifying cell growth factors from natural products cannot be ignored. Although collagen is relatively readily available, its use has recently been restricted as a potential source of mad cow disease. In other words, at present we cannot deny the danger that mad cow disease will spread to pigs,
This is because the possibility that mad cow disease factor is mixed in collagen extracted and prepared from pig skin components cannot be denied.

[0007] Accordingly, the present invention seeks to provide a cell culture bed substrate for a new and effective animal cell culture instead of collagen. INDUSTRIAL APPLICABILITY The present invention is intended to directly contribute to the cultivation of animal cells on a novel cell culture bed substrate, and is intended to produce useful substances from the animal cells, as long as the cells have a uniform genetic shape and properties. Cell cloning technology, which is a basic technology for growing E. coli, is important, and is related to the wide cloning and collection of various animal cells.

[0008] Insect cells are extremely promising as a substance production system for producing useful physiologically active substances by animal cell culture. In particular, as many as 300 or more nuclear polyhedra (NPV) viruses have been isolated from lepidopteran insects. The NPV virus inoculated into insect cells specifically infects the host insect and rapidly grows, and can be widely used as a microorganism for controlling natural pests. The NPV virus is used to control pests such as P. cucurbita.) In addition, it can be effectively used for purposes other than the control of natural enemy pests.

A mouse-type monoclonal antibody, which is a typical example of a useful substance produced in animal cells, is produced and secreted from a hybridoma grown in mouse ascites. However, not all hybridomas can grow well in mouse ascites, and even if they can grow, purification of monoclonal antibodies from the ascites involves certain difficulties. Therefore, various culture methods and culture solutions for culturing hybridomas, which are planktonic cells, in an artificial environment have been developed. On the other hand, as an example of a useful animal cell exhibiting adhesion to a substrate, a cancer cell SW-1116 derived from human colon cancer is a cancer-associated antigen C
It produces A1-9 and carcinoembryonic antigen CEA and is widely used for diagnosis of colon cancer and liver cancer.

[0010] The present invention relates to a method for preparing an animal cell including an insect cell as described above, wherein the general property is to adhere and proliferate according to the degree of adherence when the cell is adherent and to float when the cell is floating. Seeks to provide a cell culture bed for providing adhesion and preferably for attachment and growth.
Thus, the function of the cell culture bed of the present invention to selectively adhere animal cells in accordance with the degree of adherence of animal cells is also used for separation of adherent cells and planktonic cells. be able to.

[0011]

SUMMARY OF THE INVENTION In order to achieve the above object, the present invention relates to a silk protein selected according to the type of animal cell to be cultured, and comprising only one of silk fibroin and silk sericin. A cell culture bed base material for growing animal cells, comprising a membrane body containing, or both in a specific ratio, adhered to a substrate of the cell culture bed.

The above structure aims at establishing a technique by which the present inventors can control the adhesion of useful cells to the surface of a cell culture bed by changing the composition of the cell culture bed substrate. This is obtained as a result of conducting an experiment of selectively selecting one or both of silk fibroin and silk sericin on the substrate surface in a layered manner. That is, the floating cells can be reliably attached by appropriate selection of the silk protein,
It has been found that various cells can proliferate in large quantities.

[0013] The present invention also provides a method of selecting at least two types of layer materials from a mixture of silk fibroin and silk sericin and a mixture of both at a specific ratio, and making each layer water-insoluble in a cell culture bed substrate. This is a cell culture bed substrate for animal cell growth composed of those adhered in layers. .

The above-mentioned constitution has been established as a result of the present inventors examining the relationship between the method for preparing the cell culture bed base material and the cell adhesion and proliferation. That is, regardless of whether it is silk fibroin or silk sericin, it is applied as an aqueous solution when coating it on the surface of the cell culture bed substrate, and this is used as a base for the culture bed surface or another coating material. It was found that the crystallization must be promoted to make it insoluble in water when it is made insoluble. Such a water insolubilization treatment of the silk protein is performed by contacting with an alcohol or an aldehyde, or by irradiating with ultraviolet rays.

The silk fibroin and silk sericin used in the construction of the cell culture bed substrate of the present invention are preferably silk proteins obtained by sterilizing in an autoclave and then dialysis with distilled water. As a method for preparing a cell culture bed substrate, a sterile silk protein aqueous solution is brought into contact with the inner surface of a cell culture container, the silk protein aqueous solution remaining on the inner surface of the container is dried, and further subjected to a water insolubilization treatment to form a silk coating film. It basically includes steps.

A typical cell culture bed substrate according to the present invention is:
It is produced by coating a water-insoluble silk protein on the culture bed in layers. Although the material and shape of the cell culture bed carrier are not specified, the material may be, for example, polystyrene or glass, which is a polymer of styrene, and the shape (form) may be petri dish, fiber mass, cloth, tube, particle, or the like. Applied.

As the raw silk protein, silk fibroin and silk sericin derived from silkworm or wild silkworm are used. These can be obtained from liquid silk proteins in the silk glands of the silkworm, or cocoons, silk or silk products solubilized with a concentrated solution of a neutral salt such as LiBr or CaCl ₂ .

In preparing a cell culture bed substrate, first, silk fibroin (or silk sericin) is directly coated and insolubilized on the substrate surface as a first layer, and then silk sericin (or silk fibroin) is applied on the first layer. And insolubilized.
The specific procedure is as follows. An aqueous solution of silk fibroin (or silk sericin) having a concentration of 2% or less, preferably 0.1 to 0.3% is placed in a cell culture bed container, and left at room temperature for 3 to 30 minutes.
The silk protein aqueous solution is removed by a decantation method, and an aqueous solution film is formed. (The coating can also be formed by a spraying method or a dipping method.) In order to uniformly form a silk film on the surface of the substrate of the cell culture bed, the substrate (the inner surface of the container) is turned upside down and 30 to 60 times. Heat to ℃ and dry. Since the silk protein immediately after drying is soluble in water, an aqueous methanol solution (50 Wt%), which is a poor solvent for the silk protein, is added and left for 20 to 30 minutes. By forming a second layer of silk sericin (or silk fibroin) in a similar procedure on the first layer of silk fibroin (or silk sericin) thus formed, a typical cell culture bed container of the present invention is formed. Prepared.

The crystallinity of the silk fibroin or silk sericin coated on the culture bed carrier, and in some cases, a mixture of the two, is determined as a physical quantity indicating how much solubility remains or is insolubilized by the insolubilization treatment as described above. As determined by X-ray diffraction measurement, the crystallinity was 13, 9, and 1 respectively.
1%. In the present invention, the required crystallinity of the coating substance is at least 8%, preferably at least 11%.
The mixing ratio of silk fibroin and silk sericin is arbitrary, and typically 50/50 (weight ratio) is employed.

The cell culture bed base material thus prepared relatively simply can be used as a culture bed for animal cell growth more efficiently than a conventional cell culture bed base material or more efficiently. Not only cells but also buoyant cells can be grown and cultured by changing the buoyancy to adherence.

For example, Antheraea eucalipti cells derived from wild silkworm having poor adhesion to the cell culture bed substrate and Bombyx mori (SPC-Bm36) cells which are said to be derived from floating silkworm are styrene and the like. Although the adhesion to the synthetic polymer substrate is poor, it adheres firmly to the cell culture bed substrate of the present invention, and the proliferation is extremely good. Also, according to the difference in cell adhesion, the composition ratio of the cell culture bed substrate is appropriately adjusted,
It is also possible to control cell adhesion and proliferation.
Such control of cell adhesion and proliferation can be performed depending on the insolubilization or immobilization treatment of the cell culture bed substrate.

Therefore, when an aqueous solution to which a plurality of cells including cells having different genetic characteristics are added is applied to the cell culture bed of the present invention in which the adherence is controlled, the cell culture has a specific adherence (adsorption ability) to the adherence (adsorption ability). Cell sorting operation in which specific cells are strongly attached and other cells are removed together with the culture solution by a decantation method or the like. In addition, cells that are indistinguishable from the difference in appearance can also be separated from the difference in their adherence or adhesiveness.In this respect, a large amount of recombinant cells can be used as interferon, erthropoietin, tissue plasminogen activator ( It is considered that the cell culture bed substrate of the present invention plays a large role in growing useful substances such as TPA).

As the cells applicable to the cell culture bed substrate of the present invention, there are a wide range of cells such as primary cultures of various animal tissues and established cells derived from animals. Specific examples of such cells include Chinese hamster ovarian cancer cells (CHO), monkey kidney cancer cells (COS), human uterine cancer cells (Hela), and myeloid cells of humans, mice, rats, and the like. Can be targeted for any cancer cell.

Useful substances produced in connection with culturing animal cells include, in addition to the specific substances produced by the animal cells,
There are proteins and peptides with a genetic code incorporated from the outside, and these useful substances include peptide hormones, cytokines, transport proteins and the like. Therefore, the development of the novel culture system ultimately intended by the present invention can be generally applied to the production of these functional proteins involving the culture of animal cells.

[0025]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention and verification examples will be described in detail.

Example 1 Silk fibroin fiber was treated with 9.5 M LiBr at 60 ° C. 30
After dissolving the solution, the solution was placed in a dialysis membrane made of cellulose and sufficiently replaced with tap water to prepare an aqueous solution of silk fibroin having a concentration of 3.36 ml / dl as a mother liquor. This aqueous solution was used as a mother liquor, and 3.36 ml / dl of the solution itself was used.
Culture experiments of various cells were carried out by adding water to two types of 0.40 ml / dl. These silk fibroin aqueous solutions were sterilized by an autoclave, injected into 6 wells of a 12 well plate as a cell culture vessel,
After standing for a minute, the solution was substantially removed by a decantation method, the aqueous solution applied to the inner surface of the well was sufficiently dried, and further insolubilized by ultraviolet irradiation, that is, a crystal-immobilized silk protein film was formed. When the aqueous solution after sterilization is coated on the inner surface of the well by spraying or dipping, it is dried and insolubilized thereafter. In the plate, 6 wells having the silk protein film on the inner surface and 6 wells having no silk protein film but coated with collagen instead were used as a test section and a control section, respectively. 0.7 ml of a cell culture solution obtained by inoculating the cells at 1 × 10 ⁵ cells / ml into the medium, maintaining the temperature at 27 ° C. for 5 days, and then culturing each of the cultured cells And its viability was measured.

The insect-derived cells / medium used were (a) Sf9
/ IPl-41 (10% FBS), (b) Sf9 / Sf900II, (c) Bm N4 / IPl-
4 (10% FBS), (d) High five / EX-CEll400, (e) High f
Table 1 summarizes the measured cell concentrations and the cell viability after cell proliferation of the five types of iveSf900 / II0.

[0028]

[Table 1]

According to the above example, there is a slight difference between the experimental groups with the two silk protein concentrations of 0.4 ml / dl and 3.36 ml / dl. The results show that the number of cultures was almost equal to or greater than that of the control without the silk protein membrane. Therefore, it should be noted that the silk protein exerts an effect almost equal to or higher than that of collagen, and that these cells are derived from insects which have not been applied as conventionally difficult to culture.

Example 2 A middle thread gland containing a large amount of liquid silk protein was taken out from the mature silkworms of 25 silkworms (No. 137 × No. 137), washed thoroughly with distilled water, and then tipped with tweezers. Gland cells were removed. 35 g of the obtained liquid silk protein was placed in 40 ml of distilled water, and the dispersion time was changed to separately prepare silk sericin having a pH of about 6.8 and a silk fibroin aqueous solution. When the dispersion time was 30 minutes, a silk sericin solution was prepared, and when the dispersion time was 4 hours, a silk fibroin solution was prepared. Both sample solutions were added to about
The solution concentrated to 8% was used as a stock solution. In addition, an equal volume mixed solution of a silk fibroin solution and a silk sericin solution was prepared. Of these stock solutions, for example, sterilization of silk fibroin aqueous solution was performed as follows. After dissolving the silk fibroin fiber with 9.5 M LiBr at 60 ° C. for 30 minutes,
The aqueous silk fibroin solution containing the iBr solution is filtered through paper filter paper. This was packed in a cellulose dialysis membrane, and both ends were sealed with sewing threads. This was put in a container containing distilled water, and the whole container was put into an autoclave at 120 ° C. for 20 minutes.
Sterilization was performed. After the autoclave treatment, the dialysis treatment was performed while stirring the cellulose dialysis membrane in distilled water at a temperature of 5 ° C for 2 days. After dialysis, the lid of the cellulose dialysis membrane is opened in a clean bench, and the sterilized silk fibroin solution is transferred to an aseptic container to prepare for the next use. Sterilization of the silk sericin aqueous solution or the like is performed in the same manner. Thereafter, unless otherwise specified, experiments were performed using the sterilized aqueous solution of silk fibroin or silk sericin aqueous solution thus prepared.

Each of the sterilized aqueous solutions was applied to a polystyrene Petri dish (35φ × 10 mm) (Becton Dickins
on labware, Faclon 1018).
After standing for 0 minutes, the liquid phase was removed by a decantation method, and dried at 25 ° C. to cover the bottom of the Petri dish. The Petri dish washed with an aqueous solution of 10 to 15 mM sodium phosphate and distilled water and dried with a drier was treated with methanol and irradiated with ultraviolet light to fix the coating material on the bottom of the Petri dish. For the methanol treatment, a method of immersing in 50% methanol by weight for 5 minutes was employed. UV irradiation treatment is performed under a nitrogen stream under an ultraviolet lamp (National germicidal lamp, Gl
-15W) for 30 minutes under 50 cm.

In this example, cultured cells derived from tussah silkworm, Antheraea eucalypti cells (Ae cells for short)
Was examined for growth status and adherence to the cell culture bed. For the culture of the cells, fetal bovine serum and silkworm serum were used for 5 times each.
% Grace's medium. As described above, 2 × 10 Ae cultured cells were seeded on 1 ml of the medium using the Petri dish after the coating material was immobilized. It was left still in an incubator at a carbon dioxide concentration of 5% and 26 ° C., and after 65 hours,
The attachment state of the Ae cultured cells was examined. The bottom surface of the cell culture vessel was gently rocked in the field of view of an inverted microscope, and the state of attachment of the cells was evaluated from the floating state of the cells. Furthermore, in order to examine the cell culture efficiency, the silk protein cell culture bed container with the cells attached was placed in an ultrasonic pipette washer (300 W, 26 kHz),
Ultrasonic irradiation was performed at 25 ° C. for 15 seconds, the cells on the bottom of the culture vessel were detached by pipetting, and the number of cells in the culture solution was counted using a hemocytometer. Table 2 shows the obtained results. In the table, F indicates silkworm silk fibroin, S indicates silkworm sericin, M in parentheses indicates insolubilization by 50% by weight methanol treatment, and UV indicates insolubilization by ultraviolet irradiation treatment. Further, as a comparative example, the state of cell attachment by the coating F ′ formed from the silk fibroin aqueous solution that was not sterilized is shown in the bottom row of the table. F ′ used in the following examples also has this meaning. Such a comparison between F 'and F (M), F (UV) indicates that sterilization of the silk protein did not significantly alter cell adhesion, but improved the number of proliferation.

[0033]

[Table 2]

According to this example, the cell proliferation number is not much different from that of the control group, but the adhesion between the surface of the cell culture vessel and the insect cells is S + F (UV), F (UV), AF (M ).
In general, it can be seen that the material of the present invention is effective for the growth of insect cells having a weak adhesive force.

Example 3 Silkworm reared with fresh mulberry leaves (No. 137 × No. 137) 2
From the five mature silkworms, the middle gland containing a large amount of liquid silk protein was taken out. After thoroughly washing with distilled water, the silk gland cells were removed with tweezers to prepare a gel of silk protein made of liquid silk fibroin on the inside and liquid silk sericin on the outside. 35 g of this gel-state silk protein is placed in 40 ml of distilled water, and the dispersion exposed to distilled water for 45 minutes and the dispersion exposed to distilled water for 5 hours become pure silk fibroin or silk sericin aqueous solution, respectively. This silk fibroin and silk sericin were used alone to coat a commercially available cell culture bed surface, or one was first attached to the cell culture bed surface and insolubilized,
The other material was coated and adhered thereon to perform insolubilization treatment, and the surface of a commercially available culture vessel was coated in a layer to form a cell culture bed substrate.

In preparing the substrate, the concentration of these silk protein aqueous solutions was 0.6% by weight. A silk fibroin aqueous solution, a silk sericin aqueous solution, and a mixed solution of an equal amount of silk fibroin and silk sericin at this concentration were each added to a polystyrene Petri dish (35 mmφ × 10 mm) (Bect
on Dickinson labware, Faclon 1018)
After standing at 25 ° C. for 10 minutes, the sample solution was removed by a decantation method, and dried at 40 ° C. for 2 hours. The various proteins immediately after being coated with the various proteins in a different manner are water-soluble. Therefore, 25 ° C as water-insoluble treatment
Is filled in a polystyrene petri dish coated with silk protein, and left for 20 minutes. After the standing, the methanol aqueous solution is removed from the Petri dish by a decantation method, and dried with a dryer at 80 ° C.

Immediately before the cell culture experiment, the Petri dish was placed on an ultraviolet lamp (National Sterilization Lamp, Gl-15W).
It was placed under 0 cm and irradiated with ultraviolet light for 10 minutes under a nitrogen stream.

Here, the growth state of cultured cells (Ae cells) derived from wild silkworm and their adhesion to the cell culture bed were examined. Grace medium containing 5% each of fetal calf serum and heated serum of silkworm body fluid was used for culturing the cells. As described above, the Ae culture cells (8.0 × 10 ⁴ cells / ml) on day 3 of the passage were seeded in 1 ml of the medium using the Petri dishes after the coating material was immobilized. After standing still in an incubator at a carbon dioxide concentration of 5% and 26 ° C. for 65 hours, the adhesion state of the Ae cultured cells to the culture bed and the amount of cell proliferation were examined. Table 3 shows the obtained results. Cell proliferation was determined by measuring the average number of cells per ml of medium. In addition, the symbols shown in the following table including the reconfirmation mean the following.

(1) Culture bed substrate F: silkworm silk fibroin AF: tussah silk fibroin S: silkworm silk sericin F / S: silkworm silk fibroin is first coated on the cell culture bed surface and insolubilized Thereafter, a cell culture bed base material further coated with silkworm silk sericin on its surface and subjected to insolubilization treatment. S / F: A cell culture bed base material in which the silkworm sericin is first coated on the cell culture bed surface and insolubilized, and then the silkworm silk fibroin is further coated on the cell culture surface and insolubilized. A cell culture bed substrate coated with AF / S tussah silk fibroin on the surface of the cell culture bed and insolubilized, and then coated with silkworm silk sericin on the surface and subjected to the insolubilization treatment. F + S A cell culture bed substrate obtained by mixing the equivalent solution of silkworm silk fibroin and silkworm silk sericin on the cell culture bed surface and insolubilizing the mixture.

(2) Attachment state of cells on the culture bed (evaluated by the four-step method)-... not adhered and fixed + ... slightly adhered and fixed ++ ... +++ ・ ・ Very well attached and fixed

[0041]

[Table 3]

The concentrations of Ae cells and Bm cells in the initial stage of culture were 8 × 10 ⁴ cells / ml and 2.3 × 10 ⁶ cells / ml, respectively. The cell concentration on day 3 of passage in the control container was:
They were 4.8 × 10 ⁵ and 9.4 × 10 ⁶ cell / ml, respectively.

According to the verification results of this example, it is understood that the cell culture vessel of the present invention improves the proliferation of insect cells and Ae cells. AF derived from wild silkworm was particularly effective for the proliferation of Ae cells.

Comparative Example 1 F + S in the bottom row of Table 3 was used as a comparative example in the same manner as in Example 1 except that a mixed substance of silk sericin and silk fibroin was used as a cell culture bed base material. Was prepared, and an experiment was performed to adhere and grow the Ae culture cells. It can be seen that the growth state is lower than that of the stratified film F / S or S / F.

Example 4 Bombyx mori (SPC Bm 36) (Quiot, sericologia, 22, 25 (198
Using the cultured cells of 2)), the adhesion state of the cultured cells in the culture bed composed of various culture bed base materials, the number of cells cultured in the culture solution, and the adhesion were evaluated in the same manner as in Example 1. Table 4 shows the measurement results.

[0046]

[Table 4]

This example is a result of a culture experiment of SPC Bm 36 insect cells, and it can be seen that the adherence ratio is higher in the experimental plot than in the control plot, and the growth state is better.

Comparative Example 2 F + S in the bottom row of Table 4 was prepared as a comparative example by preparing a cell culture in the same manner as in Example 1 by using a mixture of silk sericin and silk fibroin as a cell culture bed base material. Then, an experiment for adherent growth of SPCBm cultured cells was performed.

In this example, a base material was prepared for comparing collagen with silk protein or examining the effect of collagen. For this, first, a collagen solution (0.3%, pH 3.0) for tissue culture (manufactured by Koken Co., Ltd.) is poured into a Petri dish, and after 10 minutes, the collagen solution is removed by a decantation method. Lightly dried. Next, a phosphate buffer of pH 7.4 is added to bring the surface of the collagen coating into a swollen state, and then the above-mentioned silk fibroin solution or silk sericin solution is added, and the mixture is left at 5 ° C. for 30 minutes.
The silk protein aqueous solution was removed and dried to prepare a silk fibroin / collagen or silk sericin / collagen culture bed coating. (Note that the boundary between the collagen layer and the silk protein layer is mixed with each other, and the range in which the two substances coexist varies depending on the conditions, and in some cases, may exhibit a single layer.) Immobilization treatment was performed by irradiation or immersion in methanol. Table 5 shows the results of measuring the number of cells attached to the cell culture bed of this example in relation to such a collagen preparation substrate.
In Table 5, C + S represents a layered film of collagen and silk sericin, and C represents a collagen film.

[0050]

[Table 5]

Example 5 Using an insect culture cell (Ae SPC Bm 36), clone NP
The V virus mass propagation experiment was performed as follows. The pre-evaluation of the infection rate of the NPV virus to these cells was 7.9%. This was cloned by the limiting dilution method to select cells highly sensitive to NPV virus. Next, the NPV viruses hypersensitive SPC Bm36 cells 10 ⁶
The cell suspension diluted to cells / ml is placed in a culture vessel made of the cell culture bed substrate of the present invention, cultured at 26 ° C. for 24 hours, and then inoculated with an NPV virus diluted to a predetermined concentration.
After 6 hours, the culture solution was removed by gently tilting the cell culture vessel, and the adhesion state of the cells was evaluated when 5% Agarose containing Grace medium was gently overlaid. The evaluation results are shown at the following evaluation levels. ± The same as the amount of Falcon adhered to the culture vessel. +… Slightly better than the amount of Falcon attached to the culture vessel. ++ ・ ・ ・ Excellent than the adhesion amount of Falcon culture vessel. Table 6 shows the obtained results.

[0052]

[Table 6]

The SPC Bm 36 cells have characteristics of poor adhesion to the culture bed substrate and low susceptibility to the NPV virus. However, the SPC Bm 36 cells can be cultured in a culture vessel comprising the cell culture bed substrate of the present invention. SPC Bm 36 cells could be attached to the surface of the culture bed substrate, and as a result, highly sensitive cells could be cloned efficiently.

Plaque formation conditions The plaque portion 2 mm or more from the edge of the formed plaque was a homogeneous clone strain free of other viruses. When the virus infection rate of this clone strain was evaluated, it was 98% in SPC Bm 36 cells, indicating that the clone NPV
It became clear that virus selection became possible.

In this example, the method was applied to a culture bed substrate composed of silk protein / collagen containing silk protein prepared in the same manner as in Example 1 or Example 3, or a culture bed carrier composed of silk protein / chitin. In the same manner as in Example 1, a sample subjected to ultraviolet irradiation treatment to fix the sample surface was used.

According to this example, it was confirmed in the present invention that the number of insect cells adhered and the adhesion were improved.

Example 6 The state of culture of adherent cells on the cell culture bed of the present invention was examined. The specifications of the culture plate were as follows. 6-well flat bottom, culture area 9.2 cm ² , capacity 16 ml / well. Culture plate made of polystyrene and hydrophilically treated (Sumitomo Bak
elite, product number MS-80060, hereinafter abbreviated as MS-a), collagen-coated commercial plate (Sumitomo Bakelite
A culture experiment was carried out using a new plate (hereinafter abbreviated as MS-c) provided with a silk fibroin coat according to the present invention and a product number MS-0006K (hereinafter abbreviated as MS-b). The cells used were adherent colon cancer cells, SW1116 American Type Culture Collection [ATCC, CCCl-233]
And the medium used was RPM11640. The number of cells in the medium at the start of the culture and the number of cells in the medium after 5 days at 25 ° C. were examined. That is, the medium was removed by decantation on the 5th day of the culture, and then treated with a trypsin-EDTA solution (0.25% trypsin-1mMEDTA) to collect the cells adhered to the cell culture bed. Cell numbers were calculated using a hemocytometer.

First, 1 ml of colon cancer cells, SW1116, stored in liquid nitrogen for storage were thawed, and washed five times with 10 ml of RPMI medium containing 10% fetal bovine serum. A cell culture experiment was started using 5 ml of a cell suspension having a cell concentration of 3 × 10 ⁴ cells / ml prepared with the same culture solution. 37 ° C, CO
The cells were cultured under the conditions of ₂ , 5.0%. Cells were collected on day 5 of culture, and the number of cells was measured. Table 7 shows the obtained results.

[0059]

[Table 7]

According to this example, an epoch-making result that useful substances such as CEA obtained from insect cells can be quantified was shown.

Example 7 In the same manner as in Example 1, mouse myeloma cells were
Hybridoma KBS 29-02, a mouse 1 gGl antibody against floating human albumin prepared by cell fusion of NS-A and B cells of mouse spleen cells obtained by immunization with human albumin by the polyethylene glycol (PEG) method. Was used to evaluate the growth status in various cell incubators. Table 8 shows the obtained results.

[0062]

[Table 8]

In this example, the same results as in Example 6 were confirmed.

Example 8 In the same manner as in Example 1, the nostril cancer cells KB of the adherent cells
[ATCC, CCl-17] was used to evaluate the growth of cells in various cell incubators. Table 9 shows the obtained results. In this example, it is clear that adherent cells grow well on the cell culture bed of the present invention.

[0065]

[Table 9]

Example 9 In order to grow a large amount of hybridomas producing a monoclonal antibody, an experiment was conducted in the following manner.

Even if colon cancer and nostril cancer cells are removed from human tissues, they must be grown in large amounts because culture tissues contain little product. If it is a cancer cell, it can be determined by observing the shape and morphology of the cancer cell by microscopic observation after proliferating. By culturing and growing the cells, the cancer growth factor can be quantified. Since a small amount of the sample cannot be detected, it is necessary to increase the tissue to produce a substance (CEA, CA19-9) produced by colorectal cancer. The amount of substances produced by cancer cells is very small in the blood of human cancer patients and cannot be prepared in large amounts. Therefore, when preparing these substances in large amounts, it is important to proliferate the cancer cells. From this perspective, Carcinoembryon, a carcinoembryonic antigen produced by SW1116
It was decided to quantify ic antigen (CEA), a cancer-associated antigen, CA19-9.

In the same manner as in Example 1, adherent colon cancer cells (SW1116) were proliferated, and metabolites from the cells were cultured for 5 days in culture. The evaluation was performed using OlYDAS-120 (manufactured by Olympus). Table 10 shows the obtained results.

[0069]

[Table 10]

Example 10 Using the hybridoma KBS 29-02, the amount of MAb antibody released from the hybridoma antibody cells was measured in the same manner as in Example 4. This MAb is a monoclonal antibody against human albumin. Table 11 shows the obtained results.

[0071]

[Table 11]

From the hybridoma KBS 29-02, it was clarified that monoclonal antibody (MAb) was certainly produced. No difference is observed depending on the cell culture bed substrate. From the above, when the cells were grown on the cell culture bed coated with the silk fibroin according to the present invention, it was clarified that the cell cultures were not adversely affected, and the adherent cells efficiently adhered and proliferated. The amount of metabolites from the corresponding cells was confirmed.

[0073]

According to the present invention, since the cell culture bed base material having excellent adhesion of animal cells is used, the adherent animal cells can be efficiently proliferated. Therefore, the cell culture substrate of the present invention can be coated on the surface of a culture vessel such as a petri dish or a flask, a roller rotating culture machine, a bottle surface, a culture plate surface, a microcarrier bead surface, etc. It can be used for culturing. The operation of cell culture becomes easy, and the centrifugation (1000 rpm 5 to 10
The need for complicated medium exchange due to (1) is unnecessary, which is useful for efficient cells and passage.

[0086] In the case of cultured animal cells, excellent results were obtained particularly in experiments using hybridomas that are important in cell engineering. Therefore, it was revealed that useful substances can be produced in large quantities in the present invention in the future.

Further, in experiments using insect cells, which are considered to be important in the determination of toxic and harmful substances in the future, excellent results of good cell adhesion could be obtained. It is clear that the method can be widely used for cloning microorganisms.

The present invention can replace the hydrophilic plate or collagen plate coated with collagen which has been conventionally diversified, and is particularly useful as a new plate replacing the collagen plate. It can be used efficiently in industrial production of useful production materials to be used.

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] March 12, 1999

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction contents]

[0011]

SUMMARY OF THE INVENTION In order to achieve the above object, the present invention relates to a silk protein selected according to the type of animal cell to be cultured, and comprising only one of silk fibroin and silk sericin. At least a two-layer membrane comprising at least a specific ratio of
The water-insolubilized product is then placed on the substrate of the cell culture bed.
FIG. 1 is a diagram illustrating a configuration of a cell culture bed base material for growing animal cells, which is included as a stratified body in the present invention.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction contents]

According to the present invention, at least two kinds of layer materials are selected from a mixture of silk fibroin and silk sericin and a mixture of both in a specific ratio, and each layer is formed by the following method.
To make it water-insoluble (before the layer is applied over it)
More layered on the substrate of the cell culture bed
Constructs a cell culture bed substrate for animal cell growth,
The characteristics of each layer are exhibited in parallel. This
Conventionally, silk fibroin or silk sericin is not used.
Even if it is misaligned, coat it on the surface of the cell culture bed substrate.
Is applied as an aqueous solution.
Even if coating with a film material consisting of an aqueous solution of
There is a prejudice that a stratified structure is realized even after water insolubilization
It can be said that there was no idea to show it.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction contents]

Therefore, the above-mentioned constitution was established from the viewpoint that a stratified structure was necessary as a result of the present inventors examining the relationship between the method for preparing the cell culture bed substrate and the cell adhesion and proliferation. Things. Then, a transitional table that becomes the base of the next layer
The water insolubilization treatment of the layer is performed by alcohol or aldehyde contact,
Alternatively, crystallization of silk protein by ultraviolet irradiation
Be sent.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0015

[Correction method] Change

[Correction contents]

Each layer containing silk fibroin and silk sericin formed on the cell culture bed substrate may be in the process of preparation or preparation.
Aqueous solution to prevent bacterial contamination during use after production
In the step, it can be formed from a silk protein membrane obtained by sterilization in an autoclave and subsequent dialysis with distilled water.
Wear. Therefore, in the preferred cell culture bed substrate of the present invention,
The procedure for preparing one layer of the silk protein membrane is as follows: 1) contacting a sterilized silk protein aqueous solution with the inner surface of the cell culture container, 2) drying the silk protein aqueous solution remaining on the inner surface of the container, and 3) further performing water insolubilization treatment. Complete the silk coating film .

[Procedure amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0030

[Correction method] Change

[Correction contents]

Example 2 A middle thread gland containing a large amount of liquid silk protein was taken out from the mature silkworms of 25 silkworms (No. 137 × No. 137), washed thoroughly with distilled water, and then tipped with tweezers. Gland cells were removed. 35 g of the obtained liquid silk protein was placed in 40 ml of distilled water, and the dispersion time was changed to separately prepare silk sericin having a pH of about 6.8 and a silk fibroin aqueous solution. When the dispersion time was 30 minutes, a silk sericin solution was prepared, and when the dispersion time was 4 hours, a silk fibroin solution was prepared. Both sample solutions were added to about
The solution concentrated to 8% was used as a stock solution. In addition, an equal volume mixed solution of a silk fibroin solution and a silk sericin solution was prepared. Of these stock solutions, for example, sterilization of silk fibroin aqueous solution was performed as follows. After dissolving the silk fibroin fiber with 9.5 M LiBr at 60 ° C. for 30 minutes,
The aqueous silk fibroin solution containing the iBr solution is filtered through paper filter paper. This sealed with thread at both ends packed in cellulosic dialysis membrane, which is placed in a container with distilled water, the container for each 120 ° C., placed in autoclave in the apparatus at 20 minutes,
Sterilization was performed. After autoclave treatment were dialyzed with stirring for 2 days cellulosic dialysis membrane in a temperature 5 ° C. in distilled water. After dialysis, the lid of the cellulose dialysis membrane is opened in a clean bench, and the sterilized silk fibroin solution is transferred to an aseptic container to prepare for the next use. Sterilization of the silk sericin aqueous solution or the like is performed in the same manner. Thereafter, unless otherwise specified, experiments were performed using the sterilized aqueous solution of silk fibroin or silk sericin aqueous solution thus prepared.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Shoji Hayasaka 4-26-408-401, Matsushiro, Tsukuba, Ibaraki Pref. 72) Inventor Shigemichi Nishikawa 945-1 Tsujiide, Shimogasa-cho, Kusatsu-shi, Shiga Wake Pharmaceutical Co., Ltd. Kusatsu Center

Claims (7)

[Claims] Claims 1. A silk protein selected according to the type of animal cell to be cultured, comprising a membrane containing only one of silk fibroin and silk sericin or both at a specific ratio. A cell culture bed base material for growing animal cells, which is applied to a substrate of a cell culture bed. 2. The cell culture bed substrate according to claim 1, wherein said silk fibroin and silk sericin are silk proteins obtained by sterilizing and dialyzing against distilled water. 3. The cell culture bed substrate according to claim 1, further comprising a collagen layer below the membrane made of silk protein. 4. A method in which at least two kinds of layer materials are selected from a mixture of silk fibroin and silk sericin, and a mixture of both at a specific ratio, and each layer is coated on a substrate of a cell culture bed in a water-insoluble manner. A cell culture bed base material for animal cell growth, wherein the base material is attached. 5. The cell culture bed substrate according to claim 4, wherein said silk fibroin and silk sericin are silk proteins obtained by sterilizing and dialyzing against distilled water. 6. The method basically includes a step of contacting an inner surface of a cell culture container with a sterile silk protein aqueous solution, drying the aqueous silk protein solution remaining on the inner surface of the container, and performing a water insolubilization treatment to form a silk coating film. A method for preparing a cell culture bed base material, characterized by comprising: 7. The silk protein aqueous solution that has been brought into contact with the inner surface of the cell culture container is substantially removed by a decantation method, and thereafter the silk protein aqueous solution remaining on the inner surface of the container is dried. Preparation method as described.