JP6879504B2 - Cell culture plate and its manufacturing method, and cell culture method - Google Patents

Description

The present invention relates to a cell culture plate, a method for producing the same, and a cell culture method. In particular, the present invention relates to a method for producing a cell culture plate suitable for evaluation of cells having high cell mass forming property.

Various culture methods have been developed and put into practical use for testing and researching cells and the like. As a cell culture method, a cell culture plate called a well plate or a microplate provided with a large number of dents (wells) is used. As the cell culture plate, a plastic plate made of polyethylene, polypropylene, polystyrene, acrylic resin or the like is widely used.

On the other hand, the plastic plate has a limited number of cells that can be cultured as it is, and a culturing method that makes it easier to cultivate cells and the like constituting a living body has been proposed. In particular, instruments for cell culture for the purpose of cell mass formation and the like are disclosed.

For example, Patent Document 1 is a polymerized high internal phase emulsion polymer (poly HIPE) adapted for the setting and use of viable cell culture plastic supplies for the growth of cells, typically mammalian cells. It is intended to disclose a cell culture base material containing, and a cell culture system for analysis of cell proliferation, differentiation and function of the base material, and is a cell culture base material containing a plurality of microcellular polymer materials. The present invention discloses a base material or the like having a pore volume of the microcellular polymer material of 88% to 92%.

Further, Patent Document 2 uses a cell culture membrane (sheet) in which cultured cells expand and proliferate, further promote composition formation into cell aggregates, express functions, and have excellent antithrombotic properties. An attempt is made to disclose a three-dimensional culture dish or a culture plate, and a cell culture sheet or the like obtained by coating a porous sheet-like material with a polyamino acid urethane copolymer and collagen. ..

Further, Patent Document 3 is intended to provide a substance capable of easily adhering and proliferating cells and the like under the same conditions as in vivo, and the bottom wall of the well of the well plate has cell affinity. It discloses a three-dimensional cell culture plate or the like which is a composite membrane of a porous membrane coated with a substance and an oxygen permeable membrane, and is configured with the side of the porous membrane inside the well.

Japanese Unexamined Patent Publication No. 2009-535025 Japanese Unexamined Patent Publication No. 2008-0759898 Japanese Unexamined Patent Publication No. 2013-215152

As disclosed in Patent Documents 1 to 3, a culture plate or the like having improved cell adhesion has been provided by improving a conventional plastic plate and using collagen in combination. It has been found that when these culture plates are used for culturing cells that form a cell mass, they form a cell mass immediately after the start of cell culture.

On the other hand, conventionally, the state and physical properties of cells as a whole such as cell clusters that have been cultured and aggregated have been observed and evaluated, but in recent years, cell observation technology and analysis technology have advanced, and individual cells have been observed and evaluated. It is possible to analyze the structure of the cell, its function and physical properties. For example, detailed parameters such as individual cell shape and intracellular fluorescence signal, which are called High Content Analysis (HCA), are detected and quantified over time as necessary. Technology for analysis is beginning to be put into practical use.

Since this analysis technique such as high-content analysis enables analysis of individual cells, it is preferable that the cells to be cultured do not form a cell mass and the individual cells are separated. However, some of the conventional culture plates and the like described above are not suitable for such high-content analysis.

First, in the case of only a plastic plate, which is a typical culture plate, the biocompatibility is low, cells are not sufficiently cultured, or cells are likely to move in the culture solution as an essential medium, so the observation position can be specified. The cell adhesion to the plate was insufficient so that the cells became unstable, and the cells to be cultured spread so as to diffuse between the cells in an unclear manner, which is called stretching. In addition, the improved culture plates exemplified in Patent Documents 1 to 3 and the like form cell clusters as aggregates, which has been the object of conventional observation, and thus it is difficult to analyze them as individual cells. ..

Under such circumstances, it is an object of the present invention to provide a culture plate capable of suppressing the formation of cell clumps and capable of culturing individual cells and having cell adhesion, and a method for producing the same and a method for culturing cells.

As a result of diligent research to solve the above problems, the present inventor has found that the following invention meets the above object, and has reached the present invention.

That is, the present invention relates to the following invention.
<1> The surface layer treatment of the surface layer of the silicone rubber film is performed so that the contact angle is 50 degrees or less, and then the collagen is brought into contact with the silicone rubber film. A method for producing a cell culture plate having a laminated film in which the collagen layer is provided on a silicone rubber film.
<2> The method for producing a cell culture plate according to <1>, wherein the surface treatment is a surface treatment by irradiating the surface layer of a silicone rubber film with ultraviolet rays of 100 to 300 nm.
<3> The method for producing a cell culture plate according to <1> or <2>, wherein the thickness of the silicone rubber film is 1 mm or less.
<4> The contact of the collagen to the silicone rubber film in the contacting step is a spray of a collagen-containing liquid, and the thickness of the collagen layer of the laminated film is 10 μm or less. > The method for producing a cell culture plate according to any one of.
<5> A cell culture method for culturing cell mass-forming cells using a cell culture plate produced by the method for producing a cell culture plate according to any one of <1> to <4>.
<6> A laminated film composed of a silicone rubber film and a collagen layer having a thickness of 10 μm or less provided on at least one surface of the silicone rubber film is provided, and the collagen layer adheres to the silicone rubber film. A cell culture plate in which the collagen layer remains even after a washing test.
<7> The well-shaped cell culture plate according to <6>, which has the laminated membrane as a bottom.

According to the present invention, a cell culture plate capable of culturing individual cells and having cell adhesion, a method for producing the same, and a cell culture method are provided.

It is a schematic cross-sectional view which shows the structure of the cell culture plate of this invention. It is a schematic cross-sectional view which shows the structure of the conventional cell culture plate. It is a graph which shows the growth curve of the cell culture days and the cell number by the cell culture plate which concerns on an Example. It is an image which shows the result of staining observation of the cell cultured by the cell culture plate which concerns on Example. It is another image which shows the result of staining observation of the cell cultured by the cell culture plate which concerns on Example.

Hereinafter, embodiments of the present invention will be described in detail, but the description of the constituent elements described below is an example (representative example) of the embodiments of the present invention, and the present invention is described below unless the gist thereof is changed. It is not limited to the contents of. When the expression "-" is used in the present specification, it is used as an expression including numerical values before and after the expression.

The method for producing a cell culture plate of the present invention includes a step of surface-treating the surface layer of the silicone rubber film so that the contact angle is 50 degrees or less, and then a step of bringing collagen into contact with the silicone rubber film. The present invention relates to a method for producing a cell culture plate having a laminated film in which a layer of collagen is provided on the silicone rubber film by the contacting step. Hereinafter, in the present application, this production method may be abbreviated as "the method for producing the cell culture plate of the present invention", and the cell culture plate produced by this method may be abbreviated as "the cell culture plate of the present invention".

The cell culture plate produced by this method for producing a cell culture plate is capable of culturing individual cells and has cell adhesion. Although the detailed principle is not clear, it is considered that the following factors are related to this. First, by ensuring oxygen permeability with a silicone rubber membrane, appropriate oxygen is supplied to the cells planted on the membrane, and the cells can grow for a long period of time. In addition, the silicone rubber film and collagen, which originally has low adhesiveness, are laminated with appropriate adhesion by surface layer treatment, and cell adhesion is maintained in this collagen layer. The laminated film, which is substantially composed of a collagen layer and a silicone rubber film, is insufficient to serve as a scaffold for forming a cell mass, but is suitable for individual cells to separate.

[Cell culture plate]
The present invention provides a cell culture plate. Here, the cell culture plate according to the present invention has the above-mentioned laminated film and can have an arbitrary shape suitable for a cell culture test or the like. A typical shape suitable for the cell culture plate of the present invention is a culture plate having wells called a multi-well plate (dish). Multi-well plates include multi-well (micro) plates such as those in the 6, 12, 48, 96, 384, and 1536 well (well) formats that are substantially standardized or standardized. These are suitable shapes for the cell culture plate of the present invention because they are suitable for automatic loading and robot processing systems, and the present invention is also suitable for mechanical analysis such as high content analysis.

[Silicone rubber film]
A silicone rubber film is used for the cell culture plate of the present invention and the method for producing the same. By using a silicone rubber film, it has gas permeability such as oxygen and carbon dioxide suitable for a cell culture plate, and cells such as living cells to be cultured are stably cultured (grown) for a long period of time. Further, the silicone rubber film is also suitable because it is easy to use as a part (particularly the bottom surface) of the cell culture plate, shape adjustment, etc., and has low toxicity to the living body and excellent biocompatibility.

In order to obtain this silicone rubber film, first, a silicone rubber compound is obtained by blending various additives with a raw rubber-like high degree of polymerization dimethylpolysiloxane and fine powder silica as a reinforcing material as basic components. Then, a silicone rubber film obtained by adding a vulcanizing agent such as an organic peroxide or a catalyst to this compound and heat-curing it at the time of use is formed into a film shape to form a silicone rubber film.

In the molding step of this silicone rubber film, low molecular weight siloxane may be contained even after the vulcanization step or the like derived from the raw material or the like. Since this low-molecular-weight siloxane may be toxic to living cells, it is preferable to use a cell culture plate of the present invention in which such low-molecular-weight siloxane is reduced as much as possible. Volatilization by heating or the like is generally performed to remove the low molecular weight siloxane. Further, in the present invention, in order to further reduce the low molecular weight siloxane contained in the silicone rubber film, it is preferable to carry out the treatment under reduced pressure, and the environment under this reduced pressure is preferably equivalent to vacuum. This treatment is preferably combined and heat-treated (for example, about 80 ° C.) under reduced pressure (vacuum state), and the treatment time is 1 hour or longer, preferably 10 hours or longer, more preferably 1 day or longer. It is particularly preferable to do so. The upper limit of the treatment time may not be set because the amount of low molecular weight siloxane can be reduced as the treatment time is lengthened, but the effect may be saturated in a certain period of time or longer. Therefore, the upper limit may be 10 days or less or about 7 days.

The thickness of the layer of the silicone rubber membrane used in the method for producing a cell culture plate of the present invention is appropriately designed according to the shape of the entire cell culture plate, the specific culture target, the detailed structure of the silicone rubber, and the like. Will be done. The upper limit of the thickness of the silicone rubber film is preferably 1 mm or less. More preferably, it is 0.5 mm (500 μm) or less. If the thickness of the silicone rubber film exceeds 1 mm and becomes too thick, oxygen permeability may decrease and it may not be suitable for cell culture. Further, the thicker the thickness, the higher the relative rigidity, and the moldability as a cell culture plate may decrease. On the other hand, the lower limit of the thickness of the silicone rubber film is preferably 50 μm or more, more preferably 100 μm or more. If the silicone rubber film is too thin, mechanical properties such as toughness as a part of the cell culture plate may be insufficient.

[Wetability of silicone rubber film]
In the method for producing a cell culture plate of the present invention, the wettability of the surface layer of the silicone rubber film described above is treated so that the contact angle is 50 degrees or less. Generally, the wettability of the surface layer of the silicone rubber film is such that the contact angle is about 90 degrees, and the wettability is low by repelling water or the like. In addition, the surface can be activated to improve the wettability by means such as ultraviolet irradiation, which will be described in detail later. By contacting collagen in this state, it is possible to obtain a product in which collagen is substantially directly laminated on the silicone rubber film.

Here, even if collagen is brought into contact with the silicone rubber film while the wettability of the silicone rubber film is low (contact angle exceeds 50 degrees), the collagen does not sufficiently adhere to the silicone rubber film and is easily peeled off. This means that when the cell culture solution to be cultured is added during cell culture or the like, the cells are washed away, or when the cell culture plate is stored when not in use, it falls off.

[Wetness]
This wettability can be measured by a contact angle meter. The wettability of the surface layer of the silicone rubber film is 50 degrees or less, more preferably 40 degrees or less, and further preferably 30 degrees or less.

[Surface processing]
As partly described above, the method for producing a cell culture plate of the present invention includes a step of surface-treating the surface layer of the silicone rubber film so that the contact angle is 50 degrees or less. For this surface layer treatment, a method for directly improving the wettability as a characteristic of the surface layer of the silicone rubber film can be appropriately adopted. The surface layer treatment step is preferably a surface layer treatment by irradiating the surface layer of the silicone rubber film with ultraviolet rays of 100 to 300 nm. Particularly preferably, the surface layer treatment is performed by irradiation with ultraviolet rays of 150 to 250 nm. By irradiating with this ultraviolet ray, only the wettability can be improved without deteriorating the physical properties of the silicone rubber film as a layer.

In the case of irradiation with ultraviolet rays, the degree of irradiation can be indexed by the wettability of the silicone rubber film as described above. The irradiation amount of such wettability depends on the type of the silicone rubber film, the irradiation method, and the like, but can be 20 to 120 seconds with an ^{irradiation intensity of about 50 mW / cm 2.} Such conditions also contribute to the improvement of the production efficiency of the cell culture plate production method of the present invention. It should be noted that, by grasping the relationship with the wettability in advance from the irradiation intensity and the irradiation time, the wettability can be grasped as an assumed value in the method for producing a cell culture plate of the present invention, which is practical. The above is a concept that it is not necessary to evaluate the wettability every time, and the present invention includes manufacturing while achieving the wettability.

[collagen]
Collagen is used in the cell culture plate of the present invention and the method for producing the same. The collagen in contact with the surface layer of the predetermined wettability of the silicone rubber layer described above, is for you form collagen layer on the surface layer of the silicone rubber membrane. By having this collagen layer, the cell culture plate of the present invention strongly adheres the cells to the collagen layer, so that subsequent analysis and the like can be stably performed.

As the collagen used in the present invention, collagen as a constituent protein of various organisms can be used. For example, mammalian collagen, marine collagen (derived from salmon, etc.), synthetic collagen and the like can be used. In particular, from the viewpoint of adhesion to cells, mammalian collagen and marine collagen are preferably used. These collagens may be used alone or in combination as appropriate.

[Collagen contact]
The method for producing a cell culture plate of the present invention includes a step of contacting collagen. The contact between the silicone rubber film and collagen is performed by an arbitrary method, but the silicone rubber film is in a state of being formed in the form of a sheet or a film, and a collagen layer is provided here. For example, this method includes a solution coating method in which a collagen-containing liquid containing collagen is applied to remove excess solvent or the like by drying or the like, and a method in which the solvent is dried and removed. The solvent used as this collagen-containing liquid (solution) has excellent dispersibility of collagen such as water and alcohol, and even if a small amount remains after the solvent is removed, it has almost no effect on the cultureability of living cells. Is used. The contact of the collagen with the silicone rubber film in this contacting step is preferably spraying of a collagen-containing liquid. As will be described later, this is a method in which the collagen layer is preferably relatively thin, and it is easy to achieve such a thickness, and the total amount of the solvent to be dried and removed is small, so that the production efficiency is good. It is a method.

[Collagen layer]
The thickness of the collagen layer (collagen layer) of the laminated membrane in the cell culture plate of the present invention is preferably 10 μm or less. It is more preferably 5 μm or less, still more preferably 2 μm or less. On the other hand, the lower limit thereof is preferably 0.2 μm or more, and particularly preferably 0.3 μm or more. This is the thickness based on the fact that it was found that the thickness of the collagen layer is within such a range, so that the cells can be cultured in a single cell state and can be easily maintained in that state for a long time. This thickness can be achieved by the spraying method described above. The thickness of the collagen layer may be obtained from the difference in thickness from the silicone rubber film before and after the collagen layer is laminated, or may be obtained from the thickness obtained by observing the cross section of the laminated film with a microscope or the like. For simplicity, the thickness calculated from the amount (spray amount) of the collagen-containing liquid or the like applied to the silicone rubber film and the collagen content concentration may be obtained.

[Laminated film]
The method for producing a cell culture plate of the present invention is to produce a cell culture plate having a laminated film in which the collagen layer is provided on the silicone rubber film by the contacting step. In this laminated film, the collagen layer in direct contact with cells has a composition of a biological substance, and the silicone rubber film exhibits appropriate gas permeability such as oxygen, so that an environment similar to the environment in the living body can be obtained. It is also considered to be excellent in that it can be done. In this laminated film, the collagen layer may be uniformly provided on the entire surface of the silicone rubber film, and when the shape is a well or the like, the recess (groove) serving as the cell culture part of the well is formed. A partial collagen layer may be provided so that it is uniform only at the bottom. One major feature of the embodiment according to the present invention is that uniform surface treatment by irradiation with a predetermined ultraviolet ray and subsequent coating with a collagen solution by spraying can achieve uniform coating. As a result, a very thin thin film of collagen is formed on the oxygen permeable membrane.

Further, based on the above-mentioned findings and aspects of the present invention, the cell culture plate of the present invention comprises a silicone rubber membrane and a collagen layer having a thickness of 10 μm or less provided on at least one surface of the silicone rubber membrane. By having a laminated film and having the collagen layer adhere to the silicone rubber film, the collagen layer can be related to a cell culture plate that remains even after a water washing test. This cell culture plate is a typical cell culture plate produced by the method for producing a cell culture plate of the present invention. The adhesiveness between the silicone rubber film and the collagen layer when this laminated film is used can be evaluated by a water washing test. In this washing test, for example, the laminated film is sufficiently immersed in a container filled with water at room temperature and allowed to stand for about 1 hour, and then the collagen layer is turned downward in the container and hand-washed. It can be shaken and then dried. After this, the presence or absence of residual collagen layer is confirmed. Depending on whether or not this residue remains as a uniform collagen layer, if it is uniform, it is determined that the collagen layer remains even after the washing test.

[Other configurations]
The cell culture plate of the present invention has a laminated film in which the above-mentioned silicone rubber film and collagen layer are laminated according to predetermined requirements. For example, when the cell culture plate has a well shape, the laminated membrane is provided at the bottom of the laminated membrane so that the collagen layer faces the upper side of the groove. FIG. 1 is a schematic cross-sectional view of a cell culture plate having the laminated film of the present invention on the bottom of a typical well-shaped cell culture plate. This cell culture plate can be produced, for example, by attaching a laminated film used for the cell culture plate of the present invention to the bottom surface of a plastic well-shaped body having an open bottom. Further, this cell culture plate can be sterilized by EOG (ethylene oxide) or the like, and is appropriately subjected to a sterilization treatment or the like before being used. Note that FIG. 2 shows a general conventional cell culture plate as a reference.

[cell]
According to the cell culture plate of the present invention, cells that easily form cell clumps (cell clump-forming cells) and the like are adhered to a predetermined position in the state of separated cells for a long time. Can be cultivated. Various living cells and the like are typical cells for which such culture is required. Specific examples include stem cells, tumor cells, and primary cultured cells (liver, kidney, skin, bone, etc.).

As described above, the cell culture plate of the present invention described above is used by seeding the cells to be cultured on the collagen layer side of the cell culture plate and culturing the cells. In this seeding, the seeding can be carried out in the state of a culture solution containing the cells to be cultured. Even if the cells are seeded in a liquid state, the cells in the culture solution are adhered to the collagen layer of the cell culture plate of the present invention and cultured, which facilitates observation and the like. And, due to this property, it is suitable for use in HCA and the like for analyzing individual cells.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples unless the gist thereof is changed.

[Example 1]
Hereinafter, a typical production example of the cell culture plate of the present invention and the cell culture result using the cell culture plate will be described.

[Evaluation device]
-Wetability test The wettability was tested using a "simple contact meter DMe-210" manufactured by Kyowa Interface Science Co., Ltd. As the wettability test solution, ultrapure water was used, and the static contact angle at room temperature (20 ° C.) was measured, and this was used as the contact angle.

[Raw materials, etc.]
-Silicone rubber film (A)
SIR Co., Ltd. "C6-530" (PDMS-based silicone rubber film) was vacuum-dried at 80 ° C. for 7 days and used as the silicone rubber film (A). The silicone rubber film (A) has a thickness of 0.3 mm (300 μm).
・ Collagen (A)
Kokensha "Pig Atelocollagen" (Mammalian Collagen)
・ Solvent (for collagen spray)
"Ultrapure water" manufactured by Water
-A 96-well body and a 384-well body with an open bottom portion of the well were used. These well-shaped bodies are used as cell culture plates by providing a sheet or the like in the opening corresponding to the bottom surface. These well-shaped bodies were made of polystyrene.

[Manufacturing of cell culture plates]
(Manufacturing Example 1)
(1) The surface layer of the silicone rubber film (A) is irradiated with ultraviolet rays having a central wavelength of 172 nm (“FLAT EXCIMER EX-mini L12530-01” manufactured by Hamamatsu Photonics Co., Ltd.) (intensity: 50 mW / cm ² , irradiation time: 30 seconds). did. After irradiation, the contact angle of the surface layer of the silicone rubber film by the wettability test was 40 degrees.
(2) After that, a collagen-containing aqueous solution having a concentration of 1% by mass was prepared, and a collagen-containing solution was applied to the surface side of the silicone rubber film irradiated with ultraviolet rays so that the residual collagen thickness after drying of the collagen-containing solution was about 1 μm. Sprayed.
(3) After that, the mixture was allowed to stand at room temperature to dry and remove the solvent (water) of the collagen-containing liquid to obtain a laminated film in which a collagen film was provided on the surface layer side of the silicone rubber film irradiated with ultraviolet rays.
(4) This laminated film was attached to the bottom surface side of the well so that the collagen layer faces the upper part of the groove of the well (the bottom of the recess of the well becomes the collagen layer). In addition, this pasting was performed by crimping by pressing. As a result, the cell culture plate (A) was produced.

[Cell culture test]
Human liver cancer-derived cells "HepG2" (RIKEN BRC Cell Material Development Office-CELL BANK) were seeded on the cell culture plate (A), and a cell culture test was conducted. The seeding density was 2 × 10 ⁴ cells / well, and the seeding liquid volume was 100 μL / well. The environment at the time of culturing was allowed to stand in a _{wet state at 37 ° C. and CO 2} concentration of 5% using an incubator.

[Evaluation method of cell culture results]
"Number of cells"
Using "CellTiter (registered trademark) 96 AQueous One Solution Assay" manufactured by Promega, the number of cells was determined and cell proliferation was evaluated.
"Fluorescent staining of cells"
(Staining reagent 1) Staining of whole cells: Hoechast 33258 (Staining: blue) (manufactured by Dojin Chemical Co., Ltd.)
(Staining reagent 2) Cytoplasm of vesicles: Calcei-AM (staining: green) (manufactured by Dojin Chemical Co., Ltd.)
(Staining reagent 3) Each of photoreceptor cells: Propidium Iodide (PI) (Staining: red) (manufactured by Takara Bio Inc.)
* The cells were fluorescently stained according to the usage procedure of each staining reagent, and the state of the cells was observed by "Operetta" (manufactured by PerkinElmer).

[Conventional cell culture plate]
The cell culture plates used in the comparison test were as follows, and the cell culture results were similarly evaluated in place of the cell culture plate (A) according to the above-mentioned cell culture test method.
(Comparative Example 1) Plastic Plate
96-well plate made by VIOLAMO (96-well plate made of polystyrene resin)
(Comparative Example 2) Easy cell mass formation plate “Gas Permeable VECELL Plate --G-Plate” manufactured by Bethel Co., Ltd. In this cell culture plate, an amphipathic polymer is applied to provide an amphipathic polymer layer without surface treatment of a silicone rubber film, and then a collagen-containing solution is further applied to provide a collagen film. Is.

The number of cells and the results of fluorescence staining of cells after culturing HepG2 by the above-mentioned cell culture test are shown in FIGS. 3 to 5.

FIG. 3 shows the number of cells. The cell culture plate (A) according to the present invention showed the same number of cells as other conventional cell culture plates, and problems such as suppression of cell culture did not occur.

FIG. 4 shows that the staining observation of the cells cultured using each cell culture plate produced as 96well was performed 2 days (2 Days) and 5 days (5 Days) from the start of the culture. Example 1 (cell culture plate (A)) according to the present invention exhibited excellent cell separation at 2 days and maintained cell separation even at 5 days. On the other hand, Comparative Example 1 (plastic plate) was cultivated in a state similar to a cell mass (however, it is not considered to have a three-dimensional structure as a cell mass) at 2 days, and extended to a culture environment at 5 days. The intercellular structure became unclear. Further, in Comparative Example 2 (I Ching cell mass forming plate), the cell mass was formed at the time of 2 days and the time of 5 days.

FIG. 5 shows stain observation of cells cultured using each cell culture plate produced as 96 well and 384 well, 2 days after the start of culture (2 Days). In Example 1 according to the present invention, each cell was observed with separability in both 96 well and 384 well. On the other hand, in Comparative Example 1, a large number of cell aggregates were observed, and few cells were isolated and cultured.

According to the present invention, a cell culture plate capable of cell culture suitable for HCA (High Content Analysis) and HCS (High Content Screening) is provided, and is expected to be applied to drug discovery screening, toxicant screening, etc., and is industrially useful. Is.

Claims (5)

A step of irradiating the surface layer of a silicone rubber film having a thickness of 1 mm or less with ultraviolet rays having a wavelength of 100 to 300 nm and surface-treating the surface layer of the silicone rubber film so that the contact angle is 50 degrees or less.
Thereafter, and a step of contacting the collagen in the surface layer of the silicone rubber layer,
By said contacting step, have a layered film in which a layer of the collagen on the silicone rubber layer,
A method for producing a cell culture plate, which comprises attaching the laminated membrane as a bottom to the well-shaped opening having an opening. The cell culture plate according to claim 1, wherein the contact of the collagen with the silicone rubber film in the contacting step is a spray of a collagen-containing liquid, and the thickness of the collagen layer of the laminated film is 10 μm or less. Production method. A cell culture method for culturing cell mass-forming cells using a cell culture plate produced by the method for producing a cell culture plate according to claim 1 or 2. It has a laminated film composed of a silicone rubber film having a thickness of 1 mm or less and a collagen layer having a thickness of 10 μm or less provided on at least one surface of the silicone rubber film, and the collagen layer adheres to the silicone rubber film. in the collagen layer it is shall be left even after the water washing test,
A cell culture plate in which the laminated membrane is attached as a bottom to the well-shaped opening having an opening. The cell culture plate according to claim 4 , wherein the collagen layer of the laminated membrane is on the recess side of the well shape, and the laminated membrane has gas permeability.

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