WO2007125894A1 - 細胞培養容器の製造方法 - Google Patents
細胞培養容器の製造方法 Download PDFInfo
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- WO2007125894A1 WO2007125894A1 PCT/JP2007/058803 JP2007058803W WO2007125894A1 WO 2007125894 A1 WO2007125894 A1 WO 2007125894A1 JP 2007058803 W JP2007058803 W JP 2007058803W WO 2007125894 A1 WO2007125894 A1 WO 2007125894A1
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- cell culture
- well
- hydrophilic
- pattern
- culture container
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/20—Material Coatings
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/12—Well or multiwell plates
Definitions
- the present invention relates to a method for producing a cell culture vessel that can be suitably used in biochemical experiments, clinical experiments, drug development studies, and the like.
- Various cell culture vessels are used in the fields of biochemical experiments, clinical experiments, and drug development research. For example, many attempts have been made to form a cell adhesion suppression layer or a cell adhesion suppression layer in a pattern on a slide glass, a cover glass, or a plastic flat plate.
- a cell culture container has been proposed in which a pattern-shaped photocured product of a photosensitive composition mainly composed of a water-soluble polymer as a cell adhesion suppression layer is formed on a flat substrate (Patent Document). 1).
- Patent Document Patent Document 1
- a high-precision and highly accurate pattern can be easily obtained by using a flat substrate, such as applying a photosensitive composition to the substrate by spin coating.
- Patent Document 1 Japanese Unexamined Patent Publication No. 2005-280076 (Claim 9, paragraph numbers [0093], [0094], etc.)
- the present invention is preferred in biochemical experiments and clinical experiments' drug development research and the like. It is an object of the present invention to provide a method for producing a cell culture container that can be used appropriately. Means for solving the problem
- a first aspect of the present invention for solving the above-described problem is a concave section serving as a region for culturing cells.
- a hydrophilic photosensitive composition is applied to the bottom of the well of the cell culture vessel main body provided with a plurality of (wells) to form a coating film, and then the coating film is subjected to pattern exposure and development to an uncured region.
- the present invention provides a method for producing a cell culture vessel, wherein a cell culture vessel is obtained by removing a region to obtain a hydrophilic film having a pattern-like photocured product on the bottom surface of the well.
- a second aspect of the present invention is characterized in that the hydrophilic photosensitive composition contains a photosensitive resin having a main chain of a water-soluble polymer and a photocrosslinkable photosensitive group.
- the method for producing a cell culture container described in the first aspect is characterized in that the hydrophilic photosensitive composition contains a photosensitive resin having a main chain of a water-soluble polymer and a photocrosslinkable photosensitive group.
- a desired pattern is provided at a tip portion of the convex portion that faces the bottom surface of the well and has a convex portion that is inserted into the well.
- the cell culture container is provided with a plurality of wells that serve as cell culture regions, and a patterned cell adhesion suppression layer is formed on the bottom of the well.
- This cell culture container is advantageous in that it can be suitably used in biochemical experiments and clinical experiments, drug development studies, and the like. Brief Description of Drawings
- FIG. 1 is a diagram showing a specific example of a method for producing a cell culture container according to the present invention.
- FIG. 2 is a diagram showing a specific example of the method for producing a cell culture container according to the present invention.
- FIG. 3 is a diagram showing a specific example of a mask.
- FIG. 4 is a diagram showing a specific example of the method for producing a cell culture container according to the present invention.
- FIG. 5 is a diagram showing a specific example of the method for producing a cell culture container according to the present invention.
- FIG. 6 is a diagram showing an outline of a mask used in Example 11.
- FIG. 7 is a diagram showing the results of test examples.
- a hydrophilic photosensitive composition is applied to the bottom surface of a well of a cell culture container body provided with a plurality of concave sections (cells) serving as a cell culture region. After the coating film is formed, the cell culture is provided with a hydrophilic coating film that also has a pattern-like photocured product force on the bottom of the well by pattern exposure and development to remove the uncured region. A container is obtained.
- the cell culture vessel main body may have any number of wells.
- the number of wells that have been widely used in the past is 1 ⁇ 2, 12, 48, 96, 384, etc.
- a commercially available well (diameter: 7 mm, depth: about 12 mm) 96 well multi-well plate (length: 75 mm, width: 115 mm) can be used. .
- the material of the cell culture vessel main body is not particularly limited! However, in order to observe the behavior of cells during culture, at least the bottom surface of the cell must be colorless and transparent or nearly colorless and transparent. Is preferred. From this point of view, plastics such as vinyl chloride, polystyrene, polypropylene, and acrylic are preferred, with plastic and glass being preferred. Well Since the exposure process is used to form a hydrophilic film on the bottom surface of the surface of the cell, the side surface of the well is colored and the cell culture volume is reduced from the viewpoint of suppressing light scattering of the side surface force of the well. A vessel body is preferred.
- the cell culture vessel main body used in the present invention may have a modified surface.
- the method for modifying the surface is a known method. Examples thereof include hydrophilized polystyrene by plasma treatment of the surface and plates coated with physiologically active substances such as poly-L-lysine, laminin, fibronectin and collagen.
- the hydrophilic photosensitive composition applied to the well of the cell culture container main body preferably contains a hydrophilic photosensitive resin.
- the hydrophilic photosensitive resin is not particularly limited as long as the photocured product can suppress cell adhesion.
- the main chain skeleton is a water-soluble polymer.
- a photosensitive resin obtained by introducing a photocrosslinkable photosensitive group as a side chain or as a main chain end group into the water-soluble polymer is preferable.
- a hydrophilic photosensitive composition containing a low molecular weight photosensitive compound such as a low molecular weight photocrosslinking agent
- unreacted low molecular weight photosensitive compound remains and is eluted during subsequent cell culture. It may be a factor that inhibits natural cell behavior.
- low molecular weight photosensitive compounds can be used if there is no influence of the residue and if the cell culture system can be managed to eliminate the residue. .
- Examples of the water-soluble polymer serving as the main chain skeleton of the hydrophilic photosensitive resin include polyethylene glycol and polybulal alcohol.
- the photocrosslinkable photosensitive group introduced into the water-soluble polymer preferably has an azide group.
- Examples of the photosensitive group having an azide group include azidobenzoic acid, and those obtained by introducing them into both ends of polyethylene glycol can be used as the hydrophilic photosensitive resin.
- the photocrosslinkable photosensitive group introduced into the water-soluble polymer is preferably a photosensitive group represented by the following formula (1) from the viewpoint of ease of introduction into the water-soluble polymer.
- R is a group selected from the following formula (2)
- R is a group selected from the following formula (3)
- At least one of 1 2 1 and R has at least one azide group.
- R is a hydrogen atom, an alkyl
- an alkyl group having an acetal group, an aryl group, an aralkyl group, or a substituent containing a basic nitrogen preferably a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or an acetal group.
- R and R both have an azide group.
- R is preferably the following formula (4) and R is preferably the following formula (5).
- the method for producing such hydrophilic photosensitive resin is not particularly limited, and may be produced by a known method.
- a method for producing a hydrophilic photosensitive resin in which a photosensitive group represented by the above formula (1) is introduced into a water-soluble polymer a water-soluble polymer having an amino group at a side chain or terminal, and Bonded with this amino group to form the structure of the above formula (1) together with the amino group Let it react with the compound.
- the remaining acetal group and hydroxy group having Molecules may be reacted.
- a water-soluble polymer having an amino group! /! Is a compound that binds to an acetal compound and forms the structure of the above formula (1) together with the amino group.
- the hydrophilic photosensitive composition is usually a solution obtained by dissolving the hydrophilic photosensitive resin in a solvent.
- the solvent is not particularly limited as long as it can dissolve the hydrophilic photosensitive resin and does not damage the cell culture vessel body, but water, an organic solvent compatible with water, or a mixture thereof is preferable.
- the organic solvent compatible with water include alcohols such as ethanol and dimethyl sulfoxide. Of these, water is particularly preferred. This is because when an organic solvent is used, the cell culture vessel body may be adversely affected, or the residual solvent after photocuring may adversely affect the cells.
- the hydrophilic photosensitive composition does not inhibit the formation of a photocured product!
- additives may be included.
- the additives include mineral acids and organic acids as acids for adjusting the pH of the photosensitive composition, and sodium hydroxide, potassium hydroxide and aqueous ammonia as bases. it can.
- salt such as sodium chloride for adjusting salt strength, buffer solution such as phosphate buffer for stabilizing pH, antifoaming agent and the like can be added.
- this hydrophilic photosensitive composition is exposed to light, a photocrosslinking reaction occurs and a hydrophilic photocured product is obtained.
- the hydrophilic photosensitive composition is applied to the bottom surface of the well to form a coating film, and then the coating film is subjected to pattern exposure and development to remove the unexposed area which is an uncured area. To do.
- a cell culture vessel can be obtained in which a hydrophilic coating having a patterned photocured product force is provided on the bottom surface of the well.
- the method of applying the hydrophilic photosensitive composition to the bottom surface of the well of the cell culture container body is not particularly limited as long as it can be applied substantially uniformly to the bottom surface of the well.
- the amount of the photosensitive composition to be applied may be determined appropriately depending on the volume of the well, but a commercially available 96-well multi-well plate having 96 wells having a diameter of about 7 mm and a depth of about 12 mm as the cell culture container body.
- the coating amount is preferably 5 to 200 ⁇ L, particularly preferably 5 to 50 ⁇ L.
- the coating thickness of the hydrophilic photosensitive composition is preferably uniform, and the coating thickness is 5 ⁇ ! About 10 / z m is preferable.
- the film thickness is less than 5 nm, it is not easy to confirm whether the film is uniformly formed.
- create a film with a film thickness exceeding 10 m increase the viscosity of the solution of the photosensitive composition. This is because a problem in the coating process is likely to occur.
- heat treatment may be performed as necessary before exposure.
- the heat treatment conditions are not particularly limited as long as they do not adversely affect the cell culture container and the hydrophilic photosensitive composition, but are usually 4 to 70 ° C for about 1 minute to 24 hours, preferably 20 to 40 °. C for 5 minutes to 1 hour.
- Pattern exposure of the coating film of the hydrophilic photosensitive composition may be performed by a conventionally known pattern exposure method.
- a mask having a desired pattern is used.
- the area corresponding to the pattern of the desired photocured product transmits the light to be exposed, and other areas do not transmit the light to be exposed.
- a chromium mask in which an emulsion mask is vapor-deposited in a pattern shape on a mask body made of a material that transmits light to be exposed, for example, glass, quartz, polymethyl methacrylate, etc. it can.
- the size of the mask having a pattern may be smaller than the area of the bottom surface of the well. In this case, they are arranged independently on the coating film. It is also possible to use a mask having a convex portion inserted into the tool larger than the area of the bottom surface of the well and having a desired pattern at the tip portion facing the bottom surface of the convex wall. In this case, the tip of the mask is attached to each well, and the tip is arranged so as to be in close contact with or close to the coating film. It is larger than the area of the bottom of the well, and it is preferable to use a mask because of its productivity.
- the mask When pattern-exposing a coating film using such a mask, the mask is placed in close contact with the coating film, and a liquid layer inert to the coating film is formed on the coating film. It is preferable that the mask is disposed in close proximity, or the mask is disposed close to the coating film via a gas layer.
- the mask When the mask is brought into close contact with or close to the coating film, interference of light to be exposed is suppressed, so that the pattern exposure can be limited to a desired specific region, and the pattern-like hydrophilic cured film that is particularly excellent after development. This is because the can be formed.
- a mask When a mask is placed in this way, exposure is performed from the side opposite to the mask coating.
- a mask having a pattern is brought into close contact with or close to the surface of the cell culture container body opposite to the side on which the coating film is provided.
- the mask side force exposure may be performed.
- the arrangement method of the mask when bringing the mask into close contact with or close to the coating film is not particularly limited as long as the mask and the coating film are not damaged.
- the light source for pattern exposure of the coating film is not particularly limited as long as it is a light source capable of exposing the hydrophilic photosensitive composition.
- X-ray, electron beam, excimer laser (F, ArF, KrF laser, etc.) and solid-state ultraviolet laser are generated as light sources.
- Light source metalno, ride lamp, xenon lamp, or high-pressure mercury lamp can be used.
- the exposure energy should be set appropriately according to the structure of the photosensitive functional group and the energy of the light source used. Usually 0. ln3jZcm 2 to 5000 mjZcm 2 , especially lmjZcm 2
- post-exposure heat treatment may be performed!
- the conditions for this heat treatment may be the same as the conditions for the heat treatment appropriately performed after the photosensitive composition is applied and before exposure.
- the method of forming a patterned photocured product by removing a non-cured region by development using a developer is not particularly limited as long as it is a method capable of dissolving the coating film in the uncured region. Yes.
- a method of immersing each cell culture container body after exposure in a developer In addition, a method of applying or spraying a developer to the cell culture vessel main body can be exemplified.
- a photocured material having a good pattern can be obtained by immersing in a developer tank for 1 minute. After forming a pattern-like photocured product by development, rinsing may be performed as necessary.
- the developer used for the development is not particularly limited as long as it has a sufficient difference in solubility between the uncured region and the cured region and does not adversely affect the cell culture vessel such as denaturation.
- a solvent that can dissolve the uncured region of the photosensitive composition coating film water, an organic solvent compatible with water, a mixed solution thereof, or the like can be used.
- organic solvents compatible with water include alcohols such as ethanol, dimethyl sulfoxide, and the like. Of these, water is particularly preferred. This is because there is no influence on the cells due to the remaining organic solvent as in the solvent of the hydrophilic photosensitive composition. When these solvents are used, a pattern having no development residue can be produced.
- the concentration of the developer is not particularly limited as long as it can dissolve the uncured region.
- the developer is a mixed solution of water and ethanol. If present, the content ratio of ethanol can take any value greater than ⁇ wt% and less than 100wt%.
- the step of drying after development is not particularly limited as long as the developer can be removed, but a constant temperature dryer, a hot plate, an air dryer, or the like can be used. It is preferable to dry at a constant temperature using a constant temperature dryer.
- the drying conditions are usually 30 to 70 ° C for about 1 minute to 24 hours, preferably 30 to 40 ° C for about 3 minutes to 1 hour.
- the cell culture container produced by the production method of the present invention can be used as it is as a cell culture container for biochemical experiments, clinical experiments, drug development research, and generally a substrate with a pattern built on a flat plate is made fine.
- Necessary for cell culture the process of putting the cell-containing aqueous solution into the dish after bonding the substrate to the surface of the dish, which is a kind of culture vessel, and the culture dish containing the cell-containing aqueous solution No need to submerge in the process. Therefore, there is a problem that the compound dissolved from the adhesive adversely affects the cells, and the patterning of the submerged substrate There is no problem that the cells wrap around the back side.
- This hydrophilic film formed on the bottom surface of the well can stably maintain its structure in any of a dry state and a solution. In addition to being able to maintain its structure sufficiently in dry and humidified conditions, it can be maintained for a long period of time, for example, 1 day or more, and further 10 days, even in water at 37 ° C or in an aqueous solvent.
- the structure is maintained stably. It is important to be able to exist stably in solution, particularly in water or an organic solvent compatible with water, and in particular, the bottom of each well of the cell culture vessel is exposed to a dry state, an aqueous solution, or an organic solvent solution. This is because they are required to be resistant to all of them.
- the aqueous solvent is not particularly limited as long as it is a solution containing water.
- the aqueous solvent include alcohols such as ethanol, a mixture of water and an organic solvent compatible with water such as dimethyl sulfoxide, potassium phosphate-hydrogen hydrogen 'hydrogen phosphate-sodium water solution, sodium hydrogen carbonate' Buffers such as aqueous sodium carbonate, inorganic sodium organic salts such as sodium chloride, potassium salt, and ammonium salt, glucose, galactose, wheat sugar, starch, heparin, heparan sulfate, etc.
- Examples thereof include saccharide aqueous solutions containing monosaccharides and polysaccharides, protein aqueous solutions, DNA, RNA aqueous solutions, liquid media, and mixtures thereof.
- minerals such as clay, metal fine particles such as gold nanoparticles, polymer fine particles such as polystyrene beads and latex particles, animals, and the like may be included.
- examples thereof include cells, plant cells, microorganisms, viruses, and mixtures thereof.
- the temperature at which the cell culture vessel produced in the present invention can be used is not particularly limited as long as the cell culture vessel is not adversely affected by alteration or deformation, but it is usually from -80 ° C to 70 ° C. Particularly preferred is 20 to 40 ° C. If the temperature exceeds 70 ° C, the photosensitive group of the photosensitive resin may be decomposed and the photocured product may not exist stably.
- the region where the hydrophilic coating is provided on the bottom surface of the well that is, the cell non-adhesive region, and the bottom surface of the cell culture container are exposed. Since the exposed region, that is, the cell adhesive region, is provided, it is suitable for use in a new culture system such as a use for culturing cells in a pattern. Specifically, when a liquid medium in which cells are suspended is placed in the well of the cell culture container obtained by the present invention, Cells can be attached to regions other than the patterned hydrophilic coating on the bottom surface of the elbow. Therefore, cells can be cultured in a pattern on the bottom of the well.
- the shape of the hydrophilic coating pattern can be easily set to a desired shape such as a hole, a dot, or a stripe depending on the design of the mask.
- the cell culture container of the present invention includes a plurality of wells, cells different from one another can be cultured as necessary, and various evaluations related to cell culture can be performed. It is a good place for research and development.
- a pattern can be formed on a multi-well plate that is frequently used for cell culture on a daily basis, a multi-well plate instrument, for example, a fluorescence measuring instrument such as an immuno reader, has been introduced. And automatic medium exchange that can process a large number of multi-well plates at once can be used as it is.
- FIG. 1 (a) is a top view of the cell culture vessel main body
- Fig. 1 (b) is a cross-sectional view taken along arrow A in Fig. 1 (a)
- Fig. 2 (a) is a diagram showing the hydrophilic photosensitive composition on the bottom of the well.
- Fig. 2 (b) is a cross-sectional view taken along line A- in Fig. 2 (a)
- Fig. 3 is a cross-sectional view of the mask
- Fig. 4 is a hydrophilic photosensitive composition.
- FIG. 5 (a) is a top view of the manufactured cell culture container
- FIG. 5 (b) is A in FIG. 5 (a). — Cross sectional view.
- the cell culture vessel main body 1 has six wells 2. As shown in FIG. 2, a water-soluble photosensitive composition is applied to the bottom surface of each well 2 of the cell culture container body 1 to form a coating film 3. Next, the coating film 3 is subjected to pattern exposure using the mask 10 shown in FIG.
- the mask body 11 is made of a transparent material, and has a cylindrical convex portion 12 inserted into the well 2 as shown in FIG. 3, and the cylindrical convex portion 12 has a bottom surface of the well 2.
- the opposite tip portion 13, that is, the tip portion 13 facing the surface on which the coating film 3 is formed, has a pattern 14 in which 16 dots formed by chromium vapor deposition that do not transmit light are formed. As shown in FIG.
- the top portion 13 of the mask 10 is attached to the well 2, and the top force of the mask 10 is also exposed while the tip portion 13 is in close contact with or close to the coating layer 3.
- the exposed areas are cross-linked and cured, so unexposed areas should be developed and removed.
- a pattern 5 having dot-like holes 4 on the coating made of a photocured hydrophilic photosensitive composition on the bottom surface of the well 2 (the “patterned photocured product” in the claims) Is formed).
- Polyethylene glycol diamine manufactured by Nippon Oil & Fats Co., Ltd., number average molecular weight 1000
- photofunctional compound 4 (2— (2- (4 azidophenol) butyl) , 3-Oxazoline-5-one
- Og 2.0 times molar equivalent to the amino group of polyethylene glycol diamine
- 70 g of tetrahydrofuran (THF) are mixed and reacted at 25 ° C for 18 hours. I let you. After completion of the reaction, THF was removed by evaporation. Thereafter, 50 g of water and 50 g of ethyl acetate were added to carry out a liquid separation extraction operation.
- the obtained photosensitive resin A was subjected to 1 H-NMR measurement, and the proton peak of the polyethylene oxide methylene chain observed at 3.5 ppm and the photofunctionality observed from 6.8 ppm to 8.7 ppm.
- the compound was identified as the target compound by the proton peak of the aromatic ring derived from product 4. From the integration ratio of these peaks, the introduction ratio of photofunctional compound 4 was 95%.
- the compound was identified as the target compound by the proton peak of the aromatic ring derived from product 4. From the integration ratio of these peaks, the introduction ratio of photofunctional compound 4 was 96%.
- the obtained photosensitive resin C was subjected to 1 H-NMR measurement.
- the proton peak of the polyethylene oxide methylene chain found at 3.5 ppm and the photofunctional compound 4 found at 6.8 ppm to 8.7 ppm were observed.
- the compound was confirmed to be the target compound by the peak peak of the derived aromatic ring. From the integration ratio of these peaks, the introduction ratio of photofunctional compound 4 was 90%.
- the mixture was reacted at 60 ° C for 24 hours. Acetal-i was 97%.
- Phosphoric acid was removed by ion exchange treatment to prepare photosensitive resin F in which a photosensitive group was introduced in an amount of 0.8 mol% relative to the hydroxy group of PVA.
- Polyuric alcohol (EG 30, manufactured by Nippon Synthetic Chemical Industry Co., Ltd.) lOOg was dissolved in 700 g of water and 200 g of methanol, and the photofunctional compound synthesized according to Synthesis Example 1 of JP-A-2003-292477 ( 10 g of 3— (4-azidophenol) N— (4,4′-dimethoxybutyl) 2 -phenol-aminoamino 2-aminoamide) and 3 g of phosphoric acid were added and reacted at 60 ° C. for 24 hours. Acetal-i was 97%. Phosphoric acid was removed by ion exchange treatment to prepare photosensitive resin G in which 0.8 mol% of the photosensitive group was introduced with respect to the hydroxy group of PVA.
- THF tetrahydrofuran
- the obtained photosensitive resin I was subjected to 1 H-NMR measurement, and the proton peak of the polyethylene oxide methylene chain found at 3.5 ppm and the photofunctionality seen from 6.8 ppm to 8.7 ppm
- the compound was identified as the target compound by the proton peak of the aromatic ring derived from product 4. From the integration ratio of these peaks, mZ (m + n) was 0.048.
- the photosensitive resin A obtained in Synthesis Example 1 was mixed with water adjusted to pH 3 with hydrochloric acid so that the total weight of the solid content was as shown in Table 2.
- the obtained aqueous solution was filtered through a 0.45 m cellulose acetate membrane filter (hereinafter referred to as “filter”) to obtain photosensitive composition I-1.
- the photosensitive resin used and the total solid weight were in accordance with Table 2 or Table 3, except that pure water was used instead of water adjusted to pH 3 in the same manner as in the preparation of photosensitive composition I.
- Compositions II to IX were obtained.
- polystyrene 96-well multi-well plate flat bottom type (Sumitomo Bakelite Co., Ltd .: Trademark “Sumilon Multi-Plate 96F”, 0.32 cm 2 Z or less “Non-Coated Resin Plate” .)It was used.
- the prepared photosensitive composition 1-1 was pipetted into each well so that the coating amount was 10 L, and was present in the well. Then, leave it in a constant temperature dryer set at 60 ° C for 30 minutes. The coating film of photosensitive composition I-1 was obtained in each well.
- Example 2 The same procedure as in Example 1 was performed except that the photosensitive resin used, each coating amount and the exposure amount present in the well were in accordance with Table 2 or Table 3, and each well of the 96-well multiwell plate was used.
- a cell culture vessel was obtained in which a photocured product of a photosensitive composition having a group of dot-shaped holes corresponding to the dot pattern was provided on the bottom surface of the substrate.
- the drying conditions were 60 ° C for 1 hour, 60 ° C for 15 hours, 37 ° C for 30 minutes and 37 ° C 1 I also went with the time.
- photosensitive composition II-1 to IV-4 and 1 1 to ⁇ 1-4 is a collagen type I made of polystyrene coated with collagen type I as a base material for general-purpose well plates.
- Coating 96well plate flat bottom type manufactured by Sumitomo Bakelite Co., Ltd .: Trademark “Sumilon Celtite C-1 Plate 96F” 0.32 cm 2 Zwell.
- non-coated glass plate glass 96well plate Flat bottom type (manufactured by Nippon Sheet Glass Co., Ltd., hereinafter referred to as “non-coated glass plate”), and 96-well plate flat bottom type of aminosilane-coated polystyrene with amino groups on the surface (manufactured by Sumitomo Bakelite Co., Ltd .: Trademark) “Sumilon Celtite PL Plate 96F” 0.32 cm 2 Zwell (hereinafter referred to as “APS-coated resin plate”) The test was performed under the condition of 3.
- a polystyrene 12-well multiwell plate flat bottom type manufactured by Sumitomo Bakelite Co., Ltd .: trademark “Sumilon Multiplate 12F”, 3.6 cm 2Z Well
- Photosensitive composition ⁇ -1 prepared in Example 2 was dispensed into each well, and a coating amount of 100 L was present in each well. Thereafter, it was left in a constant temperature dryer set at 60 ° C. and left for 30 minutes to obtain a coating film of photosensitive composition -1 in each barrel.
- each well there is a 150 / zm ⁇ dot pattern (number of dots 900. Each dot is formed by chrome deposition) in a 10mm mouth (length lOmm x width 10mm).
- the applied mask material: quartz
- exposure lOOOOmjZcm 2
- Example 1 except that the mask shown in FIG. 6 was used instead of the mask with 150 / ⁇ ⁇ ⁇ dot pattern in the 4 mm mouth, and the photosensitive compositions ⁇ -1 to ⁇ -4 were used.
- the photosensitive composition having a group of dot-shaped holes corresponding to the dot pattern is provided on the bottom of each well of the 96-well multi-well plate.
- the obtained cell culture container was obtained.
- 6 (a) is a top view of the mask used in Example 11
- FIG. 6 (b) is an enlarged view of a part of the tip of the convex portion of FIG. 6 (a)
- FIG. c) is a cross-sectional view taken along arrow A- in Fig. 6 (a). As shown in Fig.
- the mask 20 is a 115 mm x 75 mm flat substrate (material: quartz) 21 on a cylinder (material: quartz) with 6 mm ⁇ and a height of 11.6 mm. It is constructed according to the arrangement of each well, and the pattern (group of dots 24 is 135, and each dot 24 is chromium-deposited). Formed by.).
- a 100 m ⁇ dot pattern (number of dots 16 9 on a 15 mm x 75 mm flat substrate (material: quartz).
- a mask material: quartz
- chrome vapor deposition was used, and the back surface force was exposed by contacting the back surface of the 96-well multiwell plate.
- a cell culture container having a photosensitive composition ⁇ -1 to ⁇ -4 photocured product having a group of dot-shaped holes corresponding to the dot pattern on the bottom of each well of a 96-well multiwell plate is obtained. It was.
- an aqueous solvent at 25 ° C. or 37 ° C. was injected into each well, and after 3 days and 21 days, The hydrophilic coating on the bottom surface was observed to investigate the shape of the hydrophilic coating and the presence or absence of peeling from the tool bottom force.
- the aqueous solvent used was pure water or a phosphate-potassium hydrogen hydrogen 'hydrogen phosphate-sodium aqueous solution (phosphate buffer, pH 7.4).
- FIGS. Figure 7 (a) shows a collagen coat. 5 ⁇ L of photosensitive composition II-1 was applied to the rate and the drying conditions were 60 ° C for 30 minutes.
- Figure 7 (b) shows 5 ⁇ L of photosensitive composition ⁇ -1 applied to the collagen-coated plate and dried.
- Fig. 7 (c) shows the condition when the condition is 37 ° C for 30 minutes.
- Fig. 7 (c) shows the amount of photosensitive composition VI-1 applied to the collagen-coated plate at 10 L, and the drying condition is 60 ° C for 30 minutes.
- the figure shows the condition after 10L of photosensitive composition VI-1 was applied to a collagen-coated plate and the drying condition was 60 ° C for 15 hours, after being immersed in a phosphate buffer solution for 21 days.
- the hydrophilic film on the bottom surface of each well did not change in surface shape before and after immersion, and the film did not peel or collapse due to swelling, and the shape 'structure was stable. From this, the hydrophilic coating provided on the bottom surface of each well in the cell culture container obtained by the production method according to the present invention has resistance and functions in a short-term to long-term culture operation. We were able to confirm that it could be maintained.
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/298,155 US20090181158A1 (en) | 2006-04-26 | 2007-04-24 | Method of Producing Cell Culture Container |
JP2008513206A JPWO2007125894A1 (ja) | 2006-04-26 | 2007-04-24 | 細胞培養容器の製造方法 |
EP07742238A EP2011857A4 (en) | 2006-04-26 | 2007-04-24 | METHOD FOR PRODUCING A CELL CULTURAL CONTAINER |
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US (1) | US20090181158A1 (ja) |
EP (1) | EP2011857A4 (ja) |
JP (1) | JPWO2007125894A1 (ja) |
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JP2009207381A (ja) * | 2008-03-03 | 2009-09-17 | Kaneka Corp | 単球捕捉用デバイス |
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JPWO2013153873A1 (ja) * | 2012-04-09 | 2015-12-17 | 東洋合成工業株式会社 | 感光性化合物、感光性樹脂及び感光性組成物 |
JP2014023508A (ja) * | 2012-07-30 | 2014-02-06 | Tokyo Ohka Kogyo Co Ltd | 細胞培養用プレートの作製方法、この作製方法で作製された細胞培養用プレート、細胞培養方法、細胞シート作製方法、細胞シート、及び感光性樹脂組成物 |
JPWO2014112633A1 (ja) * | 2013-01-18 | 2017-01-19 | 東洋合成工業株式会社 | 細胞培養用基材及び細胞培養用基材の製造方法 |
WO2014112633A1 (ja) * | 2013-01-18 | 2014-07-24 | 東洋合成工業株式会社 | 細胞培養用基材及び細胞培養用基材の製造方法 |
WO2020175211A1 (ja) * | 2019-02-27 | 2020-09-03 | 東洋製罐グループホールディングス株式会社 | 培養容器の製造方法、及び培養容器 |
JP2020137438A (ja) * | 2019-02-27 | 2020-09-03 | 東洋製罐グループホールディングス株式会社 | 培養容器の製造方法、及び培養容器 |
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JP2020167966A (ja) * | 2019-04-04 | 2020-10-15 | 大日本印刷株式会社 | 細胞培養基材の製造方法、細胞培養基材、細胞付細胞培養基材、細胞培養容器、及び細胞付細胞培養容器 |
WO2020255529A1 (ja) * | 2019-06-17 | 2020-12-24 | 東洋合成工業株式会社 | 細胞培養基板、該細胞培養基板の製造方法及び該細胞培養基板を用いたスクリーニング方法 |
WO2022210713A1 (ja) | 2021-03-30 | 2022-10-06 | 国立大学法人北海道大学 | 細胞塊形成部材、培養容器、培養細胞の生産方法、細胞塊形成部材付き培養細胞 |
Also Published As
Publication number | Publication date |
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US20090181158A1 (en) | 2009-07-16 |
JPWO2007125894A1 (ja) | 2009-09-10 |
EP2011857A1 (en) | 2009-01-07 |
TW200811286A (en) | 2008-03-01 |
KR20080109086A (ko) | 2008-12-16 |
EP2011857A4 (en) | 2012-12-26 |
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