JP2019129748A - Culture vessel - Google Patents

Abstract

To provide a culture vessel that allows a sample to be observed and analyzed accurately, while avoiding the influence of materials constituting the culture vessel.SOLUTION: The present invention provides a culture vessel at least part of which is formed from an identical phase difference member or a null phase difference member.SELECTED DRAWING: None

Description

  The present invention relates to a culture vessel, and more particularly to a cell culture vessel.

  In the field of life science, research using animal somatic cells such as iPS cells has been actively conducted. The cells are placed in a culture container (culture dish), well plate, or other culture container, along with a culture medium (liquid containing cells and other nutrients necessary for the cells) at 37 ° C for several days. To culture over a period of several weeks.

  When observing cells cultured in a medium, the outline of the cells becomes difficult to see with a normal light source. Therefore, it is customary to observe using a polarizing microscope so that the outline of the cell can be seen well.

  The polarizing microscope has a bright / dark mode, and one of the two polarizing plates is rotated at 0 ° or 90 ° with respect to the other polarizing plate around the center of the optical path. If there is a phase difference between the members to be observed, light is likely to be transmitted at at least one of the above angles. Therefore, the light source is switched and the cells are observed in a well-viewed manner.

  Moreover, when analyzing the contents of a well plate using a microplate reader, light is irradiated with respect to each well, sliding a well plate, and a measurement etc. are performed for each well (patent document 1).

JP 2009-103480 A

  In recent years, high throughput of screening has been demanded. At the same time, techniques for observing a plurality of culture levels (cell growth rate, adhesion rate, secretion concentration, etc.) at the same time are being developed. For example, when a 96-well plate is used, there are 96 culture levels, and instead of observing each one, 96 locations are photographed at the same time, and the obtained images are stratified by some algorithm to evaluate whether or not it is possible. There is a possibility that a device will be made in the future. In that case, if there are multiple phases in the observation part (for example, the bottom surface of each well), several observation parts can be observed, but some observation parts cannot be observed even if images are taken simultaneously. Happens.

  Moreover, the single use of a culture container is desired. This is because if the culture vessel is used repeatedly, there will be a problem of contamination due to contents that cannot be washed. Further, not only the culture vessel but also sensors used for parameter measurement in the culture process are required to be disposable or non-contact type sensors. Many non-contact sensors are optical. For example, a glucose sensor reads a change in optical rotation of elliptically polarized light after passing through glucose and measures a glucose concentration and the like. When such measurement is performed, an event may occur in which an accurate glucose concentration cannot be measured depending on the measurement location due to the influence of the polarization of the container.

  The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a culture container capable of accurately observing and analyzing a sample without being affected by the material constituting the culture container. And

The inventor has intensively studied to achieve the above object. As a result, it is possible to accurately measure multiple samples at the same time by forming at least the parts used for observation and measurement with the same phase difference or zero phase difference material among the parts constituting the culture vessel. As a result, the present invention has been completed.
Here, the “same phase difference” means that the optical axis angle with respect to the average optical axis direction is ± 3 ° or less. “Zero phase difference” means “same phase difference” and in-plane phase difference Re ≦ 5 nm.
The in-plane retardation Re of the film is a value represented by Re = (nx−ny) × d unless otherwise specified. Moreover, unless otherwise indicated, the thickness direction retardation Rth of the film is a value represented by Rth = {(nx + ny) / 2−nz} × d. Further, the NZ coefficient of the film is a value represented by (nx−nz) / (nx−ny) unless otherwise specified. Here, nx represents a refractive index in a direction (in-plane direction) perpendicular to the thickness direction of the film and giving the maximum refractive index. ny represents the refractive index in the in-plane direction and orthogonal to the nx direction. nz represents the refractive index in the thickness direction. d represents the thickness of the film. The measurement wavelength is 590 nm unless stated otherwise.

The experiment that led to the present invention will be described below.
Each well using a film (ZeonorFilm (registered trademark) model number: ZF14-023 manufactured by Nippon Zeon Co., Ltd.) and a 1536 well plate (made by Kojin Bio, model number: 253601) made of acrylic material and having a size of 123 mm × 81 mm × 20 mm A plate in which the bottom portion of the plate is the same retardation member was formed by bonding.
After the plate was sterilized with ethylene oxide gas, each well of the plate was seeded with CHO cells at a concentration of 5 × 10 ⁵ cells / ml, and ham F12 medium supplemented with glucose for 7 days at 37 ° C. Cultured under. Then, using a polarized light source (Nikon Instec Co., Ltd., polarizing microscope), each light passes through the polarizing plate which can be rotated 360 ° C. from the upper side of the plate by irradiating the plate with light from the lower side. Samples containing cells in wells were observed. As a comparative example, the same experiment was performed using the above 1536 well plate without film bonding. A photograph taken from the upper side of each plate is shown in FIG. 1 (invention example) and FIG. 2 (comparative example).
As shown in the photograph of FIG. 1, it can be seen that the bottom surfaces of all wells are clearly shown in the example of the present invention using the plate in which the bottom portion of each well is the same phase difference member. Therefore, all of the samples in the plate can be observed and analyzed at a time.
On the other hand, as shown in the photograph of FIG. 2, when there are a plurality of phases on the bottom surface of each well, a shadow is generated in a part of the well due to the polarization of the container, and the sample in the plate is removed. It can be seen that there are portions that can be observed and portions that cannot be observed.

The present invention has been made on the basis of the above-mentioned novel findings, and is intended to advantageously solve the above-mentioned problems. The culture vessel of the present invention has at least a part of the same phase difference member or It is formed by a zero phase difference member.
Since the parts used when observing and analyzing the sample are formed of the same phase difference member or zero phase difference member, the material constituting the culture vessel is not affected and the sample is evaluated accurately. It can be performed.
Further, when there are a plurality of members constituting the container, it is necessary to align the arrangement directions of the members in order to establish the same phase difference. For example, if the lid is disposed in the wrong direction, the configuration of the same phase difference may be lost. Therefore, it is more preferable that at least a part of the culture vessel is composed of a zero phase difference member that does not need to consider the direction of the member.
The entire culture vessel may be formed of either or both of the same phase difference member and the zero phase difference member.

In addition, as one aspect of the culture container, a culture medium holding unit having a cylindrical shape having a bottom surface and a side wall is provided, and a lid-like member that covers an upper surface of the culture medium holding unit is optionally provided, and the bottom surface, the side wall, and the It is preferable that at least two of the lid-like members are formed of a zero phase difference member or the same phase difference member.
Even if the sample is observed obliquely from the side wall to the bottom surface by forming the side wall and the bottom surface of the culture medium holding portion with a zero phase difference member or the same phase difference member, the material constituting the culture vessel is affected. The sample can be accurately evaluated without affecting In addition, when the culture container includes a lid-like member, the bottom surface of the culture medium holding part and the lid-like member are formed of a zero phase difference member or the same phase difference member, so that the sample is cultured from above the lid-like member. Even when observing the bottom surface of the holder in a direction substantially perpendicular to the horizontal surface on which the culture vessel is placed, the sample can be evaluated accurately without affecting the material that makes up the culture vessel. It can be carried out.

Moreover, as another one aspect | mode of the said culture container, it is provided with two or more cylindrical culture holding parts which have a bottom face and a side wall, Comprising: Two or more cover parts which each cover the upper surface of said two or more culture medium holding parts And at least one of the two or more side walls, the two or more bottom surfaces, and the two or more lid portions is formed of a zero phase difference member or the same phase difference member. It is preferable.
By forming the bottom surfaces of the two or more culture medium holding parts with a zero phase difference member or the same phase difference member, the sample is moved from the top to the bottom surface of the culture medium holding part, with respect to the horizontal plane on which the culture vessel is placed. Even when analyzing samples in a plurality of culture medium holding portions simultaneously in a substantially vertical direction, the sample can be evaluated with high accuracy without the influence of the material constituting the culture vessel. In addition, by forming the respective side walls of the two or more culture medium holding portions with a zero phase difference member or the same phase difference member, the sample is viewed from the side, that is, in a direction substantially parallel to the horizontal plane on which the culture vessel is placed. In addition, even when analyzing samples in a plurality of culture medium holding units at the same time, the sample can be evaluated with high accuracy without the influence of the material constituting the culture vessel. Further, in addition to the bottom surface of the medium holding part, by forming two or more lid parts with a zero phase difference member or the same phase difference member, the culture container is moved from the top of the lid part to the bottom surface of the medium holding part. Even when analyzing samples in a plurality of culture medium holding parts at the same time in a direction substantially perpendicular to the horizontal surface, it is possible to accurately evaluate the sample without affecting the material constituting the culture vessel. it can.

Yet another aspect of the culture vessel of the present invention includes a plate-like member having a channel formed on the surface thereof, and a lid-like member that covers the channel, wherein the channel and the lid-like member are at the zero position. Preferably, the phase difference member or the same phase difference member is used.
The culture vessel is placed on the plate-like member flow path from the top of the lid-like member by forming the plate-like member flow passage and the lid-like member with a zero phase difference member or the same phase difference member. Even when observed in a direction substantially perpendicular to the horizontal plane, the sample can be evaluated with high accuracy without being affected by the material constituting the culture vessel.

As still another aspect of the culture vessel of the present invention, a lid-like member having a plate-like member having two or more channels formed on the surface and having two or more lids covering each of the two or more channels is provided. It is preferable that any member is provided, and at least two or more flow paths are formed of a zero phase difference member or the same phase difference member.
By forming two or more flow paths with a zero phase difference member or the same phase difference member, a plurality of samples are simultaneously transferred from the top to the flow path in a direction substantially perpendicular to the horizontal plane on which the culture vessel is placed. Even when analyzing the sample in the channel, the sample can be evaluated with high accuracy without the influence of the material constituting the culture vessel. Further, in addition to the flow path of the culture medium holding part, by forming two or more lid parts with a zero phase difference member or the same phase difference member, the sample is transferred from the top of the lid part to the bottom surface of the flow path. Even when analyzing samples in multiple channels at the same time in a direction substantially perpendicular to the horizontal surface, it is possible to accurately evaluate the sample without affecting the materials that make up the culture vessel. it can.

  The culture container is preferably a sheet member that is bonded to the plate member so that the lid member covers the flow path. By using the lid-like member as a sheet member, the light transmittance is increased and observation becomes easy. Moreover, since it is a sheet form, it can manufacture efficiently compared with the case where a lid | cover is formed by injection molding.

  In the culture container of the above aspect, the zero phase difference member or the same phase difference member is preferably a cycloolefin polymer. This is because an excellent effect that the phase difference can be made uniform with high accuracy can be obtained. Moreover, it is superior to other materials from the viewpoints of excellent transparency, low autofluorescence, no cytotoxicity, and high floating cell culture efficiency due to low protein adsorption.

  According to the present invention, it is possible to accurately observe and analyze a sample without affecting the material constituting the culture vessel.

FIG. 1 is a polarized light micrograph of a 1536 well plate used for cell culture where the bottom of each well is the same retardation member. FIG. 2 is a polarization micrograph of a 1536 well plate used for cell culture and having multiple phases on the bottom of each well. FIG. 3 is a polarized micrograph of the 96-well plate used for cell culture in Example 1. FIG. 4 is a polarizing micrograph of a 96-well plate used for cell culture in Example 2. FIG. 5 is a polarizing micrograph of a 96-well plate used for cell culture in Comparative Example 1. FIG. 6 is a polarized micrograph of the microchannel chip used for cell culture in Example 12. FIG. 7 is a polarized micrograph of the culture bag used for cell culture in Example 13.

Hereinafter, embodiments of the present invention will be described in detail.
<Culture container>
The culture container of the present invention is at least partially formed of the same phase difference member or zero phase difference member.
Specifically, the surface of the culture vessel used for observing or analyzing the sample in the culture vessel may be formed of the same phase difference member or zero phase difference member.

  For example, when simultaneously observing or analyzing a plurality of samples from the top of the culture container to the bottom surface of the culture medium holding portion, that is, in a direction substantially perpendicular to the horizontal plane on which the culture container is placed, at least each culture medium The bottom surface of the holding part only needs to be formed of the same phase difference member or a zero phase difference member. By doing in this way, even when analyzing a plurality of samples at the same time, it is possible to perform observation and analysis with high accuracy without the influence of the material constituting the culture vessel.

  In the case where the culture container further includes a lid-like member and the sample is observed or analyzed from above the lid-like member, at least the lid-like member is the same phase difference member or zero phase difference member in addition to the bottom surface of the culture medium holding unit. What is necessary is just to be formed. When the culture container has a plurality of culture medium holding parts, or when observing or analyzing a plurality of culture containers side by side, in addition to the bottom surface of each culture medium holding part, at least the lid that covers each culture medium holding part has the same phase difference. What is necessary is just to be formed with the member or the zero phase difference member. By doing in this way, even when analyzing a plurality of samples at the same time, it is possible to perform observation and analysis with high accuracy without the influence of the material constituting the culture vessel.

  For example, when observing or analyzing a sample inside the culture medium holding portion from the side, that is, in a direction substantially parallel to the horizontal plane on which the culture vessel is placed, at least the side wall has the same phase difference member or zero. What is necessary is just to be formed with the phase difference member. When the culture container has a plurality of culture medium holding parts, or when observing or analyzing a plurality of culture containers side by side, at least the side walls of each culture medium holding part are formed of the same phase difference member or zero phase difference member Good. By doing in this way, even when analyzing a plurality of samples at the same time, it is possible to perform observation and analysis with high accuracy without the influence of the material constituting the culture vessel.

  In addition, when observing or analyzing the sample inside the culture medium holding portion obliquely from the side wall to the bottom surface, by forming the side wall and the bottom surface of the culture medium holding portion with the same phase difference member or zero phase difference member, The sample can be observed and analyzed with high accuracy without the influence of the material constituting the culture vessel. When the culture container has a plurality of culture medium holding parts, or when observing or analyzing a plurality of culture containers side by side, the side wall and the bottom surface of each culture medium holding part are formed with the same phase difference member or zero phase difference member. By doing so, even when analyzing a plurality of samples at the same time, it is possible to perform observation and analysis with high accuracy without the influence of the material constituting the culture vessel.

For example, when the culture container is a plate-like member having a channel (medium holding unit) formed on the surface and further includes a lid-like member having a lid that covers the channel, the channel and the lid are provided. By forming with the same phase difference member or zero phase difference member, the material constituting the culture vessel is not affected by the material constituting the culture vessel, and the sample is accurately observed and analyzed regardless of the angle of analysis. be able to. When the plate-shaped member has a plurality of flow paths, or when observing or analyzing a plurality of plate-shaped members side by side, by forming each flow path with the same phase difference member or zero phase difference member, Even when analyzing a plurality of samples at the same time, observation and analysis can be performed with high accuracy without the influence of the material constituting the culture vessel.
The entire culture vessel may be formed of either or both of the same phase difference member and the zero phase difference member.

The material of the zero phase difference member or the same phase difference member may be any material as long as it has a transparency that allows the inside to be observed when the culture vessel is formed. For example, cycloolefin polymer, polyethylene terephthalate (PET), acrylic resin Polycarbonate can also be used, but is not limited thereto.
Of the above materials, it is most preferable to use a cycloolefin polymer. This is because the phase difference can be easily controlled and the film can be formed thin, so that it has excellent characteristics that can easily realize a low phase difference. Furthermore, it is superior to other materials from the viewpoints of high visible light permeability, no cytotoxicity, low autofluorescence, low protein adsorption and the ability to culture floating cells.
In order to make these materials into a zero phase difference member or the same phase difference member, it is necessary to anneal the injection molded product, and in the film molding, it is necessary to “thin or uniformly stretch in the same direction”.

  Here, the cycloolefin polymer is a polymer having an alicyclic structure in one or both of the main chain and the side chain. Examples include norbornene polymers, monocyclic olefin polymers, cyclic conjugated diene polymers, vinyl alicyclic hydrocarbon polymers, and hydrides thereof. Among these, from the viewpoints of transparency and moldability, a norbornene polymer is preferable, and a norbornene polymer having no polar group in the side chain is more preferable.

  Examples of the norbornene polymer include a ring-opening polymer of a monomer having a norbornene structure, a ring-opening polymer of a monomer having a norbornene structure and an arbitrary monomer, or a hydride thereof; norbornene An addition polymer of a monomer having a structure, an addition polymer of a monomer having a norbornene structure and an arbitrary monomer, or a hydride thereof.

  Here, the norbornene-based polymer is a polymer containing a monomer unit having a norbornene skeleton in an amount of 50% by mass or more, preferably 60% by mass or more based on all monomer units constituting the norbornene-based polymer. . More specifically, the norbornene polymer is obtained by polymerizing a norbornene monomer that is a monomer having a norbornene skeleton, and is obtained by ring-opening polymerization and by addition polymerization. It is divided roughly into.

Examples of the ring-opening polymer obtained by ring-opening polymerization include ring-opening polymers of norbornene monomers, ring-opening polymers of norbornene monomers and other monomers capable of ring-opening copolymerization, and these And hydrides of the above.
Examples of the polymer obtained by addition polymerization include addition polymers of norbornene monomers and addition polymers of norbornene monomers and other monomers copolymerizable therewith.
The norbornene polymers can be used alone or in combination of two or more.
Among these, a ring-opening polymer hydride of a norbornene-based monomer is preferable because the effects of the present invention are more easily obtained.

Examples of norbornene monomers that can be used for the synthesis of norbornene polymers include bicyclo [2.2.1] hept-2-ene (common name: norbornene), 5-methyl-bicyclo [2.2.1]. Hept-2-ene, 5,5-dimethyl-bicyclo [2.2.1] hept-2-ene, 5-ethyl-bicyclo [2.2.1] hept-2-ene, 5-ethylidene-bicyclo [ 2.2.1] hept-2-ene, 5-vinyl-bicyclo [2.2.1] hept-2-ene, 5-propenylbicyclo [2.2.1] hept-2-ene, 5-methoxy Carbonyl-bicyclo [2.2.1] hept-2-ene, 5-cyanobicyclo [2.2.1] hept-2-ene, 5-methyl-5-methoxycarbonyl-bicyclo [2.2.1] Bicyclic monomers such as hepta-2-ene;
Tricyclo [4.3.0 ^1,6 . 1 ^2,5 ] deca-3,7-diene (common name: dicyclopentadiene), 2-methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, 2,3-dihydroxydicyclopentadiene, etc. Monomer;
Tetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene (tetracyclododecene), tetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-methyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethylidenetetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8,9-dimethyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethyl-9-methyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethylidene-9-methyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-methyl-8-carboxymethyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 7,8-benzotricyclo [4.3.0.1 ^2,5 ] dec-3-ene (common name: methanotetrahydrofluorene: 1,4-methano-1, 4,4a, 9a-tetrahydrofluorene), 1,4-methano-8-methyl-1,4,4a, 9a-tetrahydrofluorene, 1,4-methano-8-chloro-1,4,4a, 9a -Tetracyclic monomers such as tetrahydrofluorene and 1,4-methano-8-bromo-1,4,4a, 9a-tetrahydrofluorene;
These norbornene monomers may have one or more substituents. Examples of the substituent include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.

  Other monomers capable of ring-opening copolymerization with norbornene monomers include cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, 1,5-cyclodecadiene, And monocyclic cycloolefin monomers such as 1,5,9-cyclododecatriene and 1,5,9,13-cyclohexadecatetraene.

Other monomers capable of addition copolymerization with a norbornene monomer include α-olefin monomers having 2 to 20 carbon atoms such as ethylene, propylene, 1-butene, 1-pentene and 1-hexene; cyclobutene, cyclopentene, cyclohexene, cyclooctene, tetracyclo ^{[9.2.1.0 2,10.} Cycloolefin monomers such as 0 ^3,8 ] tetradeca-3,5,7,12-tetraene (also referred to as 3a, 5,6,7a-tetrahydro-4,7-methano-1H-indene); And non-conjugated diene monomers such as 4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, and 1,7-octadiene.
Among these, as other monomers that can be addition copolymerized with norbornene monomers, α-olefin monomers are preferable, and ethylene is more preferable.
These other monomers may have one or more substituents. Examples of the substituent include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.

A ring-opening polymer of a norbornene-based monomer, or a ring-opening polymer of a norbornene-based monomer and another monomer capable of ring-opening copolymerization with a monomer component is a known ring-opening polymerization. It can be obtained by polymerization in the presence of a catalyst.
Examples of the ring-opening polymerization catalyst include a catalyst comprising a metal halide such as ruthenium or osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a metal halide or acetylacetone such as titanium, zirconium, tungsten, or molybdenum. A catalyst comprising a compound and an organoaluminum compound can be used.
The ring-opening polymer hydride of a norbornene-based monomer is usually prepared by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to the polymerization solution of the above-described ring-opening polymer, and then adding a carbon-carbon unsaturated bond. Can be obtained by hydrogenation.

An addition polymer of a norbornene monomer, or an addition polymer of a norbornene monomer and another monomer copolymerizable with the norbornene monomer, in the presence of a known addition polymerization catalyst. It can be obtained by polymerization.
As the addition polymerization catalyst, for example, a catalyst composed of a titanium, zirconium or vanadium compound and an organoaluminum compound can be used.

  There is no particular limitation on the molecular weight of the norbornene polymer, but the polyisoprene equivalent weight average molecular weight measured by gel permeation chromatography (GPC) of a cyclohexane solution (a toluene solution when the polymer does not dissolve), Usually, it is 5,000 or more, preferably 5,000 to 500,000, more preferably 8,000 to 200,000, particularly preferably 10,000 to 100,000. When the weight average molecular weight is within this range, the mechanical strength and the moldability are highly balanced, which is preferable.

The glass transition temperature of the norbornene polymer may be appropriately selected according to the purpose of use, but is usually 50 to 300 ° C, preferably 100 to 280 ° C. When the glass transition temperature is within this range, heat resistance and molding processability are highly balanced and suitable.
The glass transition temperature of the norbornene polymer is measured based on JIS K 7121.

The norbornene polymers can be used alone or in combination of two or more.
Further, as a resin component constituting the surface of the molded body, in addition to the norbornene-based polymer, optionally, a compounding agent usually used in thermoplastic resin materials, for example, a soft polymer, an antioxidant, an ultraviolet absorber, Additives such as light stabilizers, near-infrared absorbers, release agents, colorants such as dyes and pigments, plasticizers, antistatic agents, fluorescent brighteners, and the like can be added. Here, when a soft polymer is mixed with the norbornene polymer, it is usually 0.01 to 20 parts by mass, preferably 0.05 to 10 parts by mass with respect to 100 parts by mass of the norbornene polymer. Part, more preferably 0.05 to 5 parts by mass.

In addition to the norbornene polymer and the soft polymer that is one of the above-mentioned compounding agents, other polymers (hereinafter simply referred to as “other polymers”) are used as the resin component constituting the surface of the molded body. You may mix. The amount of the other polymer mixed with the norbornene polymer is usually 200 parts by mass or less, preferably 150 parts by mass or less, and more preferably 100 parts by mass or less with respect to 100 parts by mass of the norbornene polymer.
If the proportion of various compounding agents and other polymers to be blended with respect to the norbornene polymer is too large, the cells are difficult to float. Therefore, it is preferable to blend them in a range that does not impair the properties of the norbornene polymer.

The mixing method of the norbornene polymer and the compounding agent or other polymer is not particularly limited as long as the compounding agent is sufficiently dispersed in the polymer. Moreover, there is no special restriction | limiting in the order of a mixing | blending. As a blending method, for example, a method of kneading a resin in a molten state using a mixer, a uniaxial kneader, a biaxial kneader, a roll, a Brabender, an extruder, etc .; after being dissolved and dispersed in an appropriate solvent, And a method of removing the solvent by a coagulation method, a casting method, or a direct drying method.
When a biaxial kneader is used, after kneading, it is usually extruded in a rod shape in a molten state, cut into an appropriate length with a strand cutter, and pelletized in many cases.

<Method of forming culture vessel>
The method for forming the culture vessel can be arbitrarily selected according to the shape of the culture vessel. Specific examples of molding methods include injection molding, extrusion molding, cast molding, inflation molding, blow molding, vacuum molding, press molding, compression molding, rotational molding, calendar molding, rolling molding. Method, cutting molding method, spinning, and the like. These molding methods can be combined, or post-treatment such as stretching can be performed as necessary after molding.

Examples of the shape of the culture container include a dish, a plate, a microchannel chip, a bag, a tube, a scaffold, a cup, and a jar fermenter.
For example, when forming a 96 well plate in which the bottom of each well (medium holding part) is formed of a zero phase difference member or the same phase difference member, after forming an integral well plate by injection molding, glass Formed by annealing at a temperature near the transition temperature, or by forming a plate with a hole in the bottom and pasting a film with zero or the same phase difference to close the hole in the bottom To do.
When forming a 96-well plate in which the side walls of each well are formed of a zero phase difference member or the same phase difference member, after forming an integral well plate by injection molding, annealing is performed at a temperature near the glass transition temperature. It is formed by processing or by fixing a zero retardation film to a plate like a honeycomb structure.
When forming a 96-well plate in which the side wall and the bottom surface of each well are formed of a zero phase difference member or the same phase difference member, after forming an integral well plate by injection molding, a temperature around the glass transition temperature The film is formed by annealing, or by forming a side wall like a honeycomb structure and bonding the film to the bottom surface.
When forming a cover (lid member) that covers each well of a 96-well plate and has a lid formed with a zero phase difference member or the same phase difference member, an integral well plate is formed by injection molding. After that, the film is formed by annealing at a temperature near the glass transition temperature, or by bonding the film to a member made by injection molding of the outer peripheral frame of the lid.

For example, when the flow path of a plate-like member having a flow path formed on the surface thereof, such as a micro flow path chip, is formed by a zero phase difference member or the same phase difference member, injection molding is performed and an integrated well plate Then, annealing is performed at a temperature near the glass transition temperature, or a zero retardation plate is formed by extrusion.
When the sheet member (lid member) that covers the surface of the micro-channel chip is formed of a zero phase difference member or the same phase difference member, after forming an integral well plate by injection molding, the glass transition temperature It is formed by pasting a plate that has been annealed at a temperature in the vicinity so as to cover the channel, or by pasting an extruded film.

<Sterilization of culture container>
The culture container is preferably sterilized before being used for cell culture.
There are no particular restrictions on the sterilization method, heating methods such as the high-pressure steam method and dry heat method, radiation methods that irradiate radiation such as γ rays and electron beams, irradiation methods that irradiate high frequencies, and ethylene oxide gas (EOG) The method can be selected according to the shape of the culture vessel and the cells to be used from methods generally employed in the medical field, such as a gas method in which a gas is brought into contact, a filtration method using a sterilization filter, and the like. Among these, the gas method is preferable because the change in the polar state of the surface is small.

<Cell culture>
In this embodiment, the culture medium in which the cells are suspended is transferred into a culture container, and the culture container is left to be cultured.
The types of cells to be cultured include, for example, CHO cells, HEK cells, NIH cells, iPS cells, iPS-derived neurons, and the like, but are not particularly limited and can be arbitrarily selected according to the purpose.
The cell culture conditions are not particularly limited, and can be appropriately determined according to the cells to be used and the purpose. For example, the cells can be cultured using a humidified thermostat having a carbon dioxide concentration of about 5% and a constant temperature in the range of 20 ° C to 37 ° C.

The cells as described above are cultured using a liquid medium.
As the liquid medium, a medium having a pH buffering action, having an osmotic pressure suitable for cells, containing nutrient components of cells, and not toxic to cells is used.
Examples of the component that imparts a pH buffering action to the liquid medium include Tris hydrochloride, various phosphates, and various carbonates.
The osmotic pressure of the liquid medium is usually adjusted using an aqueous solution in which the concentrations of potassium ions, sodium ions, calcium ions, glucose and the like are adjusted so as to be almost the same as the osmotic pressure of the cells. Specific examples of the aqueous solution include physiological saline such as phosphate buffered saline, Tris buffered saline, and HEPES buffered saline; Ringer's solution such as lactated Ringer's solution, acetated Ringer's solution, and bicarbonated Ringer's solution; It is done.
Examples of the nutrient component of the cell include amino acids, nucleic acids, vitamins, minerals and the like.
As the liquid medium, a commercially available medium suitable for various cells can be used.

An additive can also be mix | blended with a liquid culture medium.
Additives used include peptides; minerals; metals; vitamin components; ligands, agonists and antagonists that act on cell surface receptors; nuclear receptors, ligands, agonists and antagonists; extracellular such as collagen and fivenectin Matrix: A part of the extracellular matrix or a compound that mimics the extracellular matrix; a component that acts on a protein involved in a signal transduction pathway in a cell; a component that acts on an enzyme of primary or secondary metabolism in a cell; Components that affect gene expression in the nucleus or mitochondria.
These additives can be used individually by 1 type or in combination of 2 or more types.

<Observation and analysis method>
When the culture container is a 96-well plate, a 1536-well plate, or the like and has a plurality of medium holding portions, the analysis is performed once for each plate. When the culture container has only one medium holding part, a plurality of culture containers are arranged vertically and / or horizontally, specifically, for example, eight culture containers are arranged in a horizontal row and analyzed at a time. be able to. When the culture container includes a lid-like member having a lid portion that covers the culture medium holding portion, the sample can be observed and analyzed by removing the lid-like member or from above the lid-like member.

  When the culture container is a plate-like member such as a micro-channel chip and a plurality of channels (medium holding units) are formed on the surface, the analysis is performed for each plate-like member at a time. When the plate-like member has only one flow path, a plurality of plate-like members are arranged vertically and / or horizontally, specifically, for example, four plate-like members are arranged two by two vertically and horizontally at a time. Analysis can be performed. When the plate-like member has a lid-like member such as a sheet member having a lid portion that covers the flow path, the sample is observed or analyzed by removing the lid-like member or from the top of the lid-like member. It can be carried out.

As a method for observing the sample in the culture vessel, a strain inspection device, a polarizing microscope, Cell Watcher Plus, and a cell culture observation device are used as the observation device.
As a method for analyzing the sample in the culture vessel, medium component analysis, oxygen solubility analysis, viable cell number analysis, and the like are performed using a glucose sensor, an O ₂ sensor, and a camera as an analyzer.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples.

Example 1
A COP (cycloolefin polymer) film (zero phase difference member) having a thickness of 0.021 mm and having a phase difference of zero produced by the melt extrusion method described in Example 1 of JP-A-2016-179608 is used for a 96-well plate. Cut to the size of the bottom surface (71 mm × 104 mm) and pasted into the cavity of the injection mold, and the material of “ZEONOR (registered trademark) 1060R” (manufactured by Nippon Zeon Co., Ltd.) (hereinafter referred to as COP1060R). By performing insert molding, a 96-well plate in which the film and the injection-molded product were integrated was produced (the bottom surface and the side wall were zero phase difference members).
After sterilizing the plate with EOG, each well of the plate was seeded with CHO cells at a concentration of 2 × 10 ⁵ cells / ml, and ham F12 medium supplemented with glucose for 7 days at 37 ° C., carbon dioxide Culturing was performed under conditions of a concentration of 5%. Using a polarized light source (Lukeo Co., Ltd., strain tester LSM-4100LE), light that has passed through the polarizing plate is irradiated onto the plate from the lower side, and can be rotated 360 ° C. from the upper side of the plate Samples containing cells in each well were observed. A photograph of the plate taken from above is shown in FIG.
As shown in the photograph of FIG. 3, it can be seen that the bottoms of all wells are clearly visible. Therefore, all the samples in the plate could be observed and analyzed at once.

(Example 2)
A COP film (same phase difference member) having a uniform retardation of 0.021 mm produced by the melt extrusion method described in Example 1 of JP-A-2016-179608 is the size of the bottom surface of a 96-well plate ( 71mm x 104mm), pasted into the cavity of the injection mold and insert molded with the material of COP1060R to create a 96 well plate with integrated film and injection molded product (bottom and side walls) Are the same phase difference member).
After performing sterilization treatment, cell culture and treatment using the 96-well plate in the same manner as in Example 1, the sample containing cells in each well was observed using the polarized light source. A photograph of the plate taken from above is shown in FIG.
As shown in the photograph of FIG. 4, it can be seen that the bottoms of all wells are clearly visible. Therefore, all the samples in the plate could be observed and analyzed at once.

Example 3
The COP film (zero phase difference member) having zero retardation in Example 1 above was applied with a PDMS adhesive to a 96-well plate having holes formed in the bottom of a well formed by an injection mold using the material of COP1060R. Thus, a film bottom well plate was prepared (each bottom surface was a zero phase difference member).
After performing sterilization treatment, cell culture, and treatment using the film bottom well plate in the same manner as in Example 1, the sample containing cells in each well was observed using the polarized light source.
Similar to Example 1, the bottom surfaces of all wells could be clearly imaged. Therefore, all the samples in the plate could be observed and analyzed at once.

Example 4
The COP film (same phase difference member) having a uniform phase difference of Example 2 is pasted with a PDMS adhesive on a 96-well plate having a hole formed in the bottom of a well formed by an injection mold using the material of COP1060R. Thus, a film bottom well plate was prepared (each bottom surface is the same retardation member).
After performing sterilization treatment, cell culture, and treatment using the film bottom well plate in the same manner as in Example 1, the sample containing cells in each well was observed using the polarized light source.
As in Example 2, the bottom surfaces of all wells could be clearly imaged. Therefore, all the samples in the plate could be observed and analyzed at once.

(Example 5)
A 96-well plate made of COP1060R molded by injection molding was placed in an annealing furnace at 90 ° C. for 24 hours, and a 96-well plate having a zero phase difference across the well plate was produced. Similarly, a cover (lid-shaped member) covering the 96-well plate was produced by injection molding, and placed in an annealing furnace at 90 ° C. for 24 hours to produce a 96-well plate cover having a zero phase difference across the entire cover ( The whole culture vessel is a zero phase difference member).
After sterilizing the plate and cover with EOG, each well of the plate was seeded with CHO cells at a concentration of 5 × 10 ⁵ cells / ml, and ham F12 medium supplemented with glucose for 7 days at 37 ° C. Culturing was performed under the condition of a carbon concentration of 5%. A sample containing cells in each well is passed through the polarizing plate that can be rotated 360 ° C. from the upper side of the cover that covers the plate by irradiating the plate with light from the lower side using the polarized light source. Observed.
The bottom surfaces of all wells could be clearly imaged from the top of the cover. Therefore, it was possible to observe and analyze all the samples in the plate at the same time with the lid on.

(Comparative Example 1)
A 96 well plate made of COP1060R molded by injection molding was prepared. Similarly, a cover (lid member) covering the 96-well plate was produced by injection molding.
Samples containing cells in each well from above the cover that covers the plate using the polarized light source after performing sterilization treatment, cell culture and treatment using the 96 well plate in the same manner as in Example 5. Was observed. A photograph of the plate taken from above is shown in FIG.
As shown in the photograph in FIG. 5, because the phase of the plate and cover is not controlled, there are multiple phases on the bottom of each well, and the shadow of some of the wells is affected by the polarization of the container. In the plate, there were portions where the sample could be observed and portions where the sample could not be observed. For this reason, it was not possible to observe and analyze all the samples in the plate at once.

(Example 6)
A highly stretched PET film (same phase difference member) having a uniform retardation of 0.021 mm is cut into the size (71 mm × 104 mm) of the bottom surface of a 96-well plate, and the material of COP1060R is used as in Example 1. By injection molding, a 96-well plate in which the film and the injection-molded product were integrated was created (the bottom and side walls are the same phase difference member).
After performing sterilization treatment, cell culture and treatment using the 96-well plate in the same manner as in Example 1, the sample containing cells in each well was observed using the polarized light source.
Similar to Example 1, the bottom surfaces of all wells could be clearly imaged. Therefore, all the samples in the plate could be observed and analyzed at once.

(Example 7)
A highly stretched acrylic film (same retardation member) having a uniform retardation of 0.021 mm is cut into the size (71 mm × 104 mm) of the bottom surface of a 96-well plate, and the material of COP1060R is used as in the first embodiment. By injection molding, a 96-well plate in which the film and the injection-molded product were integrated was created (the bottom and side walls are the same phase difference member).
After performing sterilization treatment, cell culture and treatment using the 96-well plate in the same manner as in Example 1, the sample containing cells in each well was observed using the polarized light source.
Similar to Example 1, the bottom surfaces of all wells could be clearly imaged. Therefore, all the samples in the plate could be observed and analyzed at once.

(Example 8)
A highly stretched polycarbonate film (same retardation member) having a uniform retardation of 0.021 mm is cut into a size (71 mm × 104 mm) of the bottom surface of a 96-well plate, and the material of COP1060R is used as in the first embodiment. By injection molding, a 96-well plate in which the film and the injection-molded product were integrated was created (the bottom and side walls are the same phase difference member).
After performing sterilization treatment, cell culture and treatment using the 96-well plate in the same manner as in Example 1, the sample containing cells in each well was observed using the polarized light source.
Similar to Example 1, the bottom surfaces of all wells could be clearly imaged. Therefore, all the samples in the plate could be observed and analyzed at once.

Example 9
A culture vessel having a lid-like member in which the COP film of Example 3 was adhered to the upper surface of the 96-well plate (made by COP1060R) of Example 3 was prepared (the bottom surface and the lid were zero phase difference members).
After performing sterilization treatment, cell culture and treatment using the 96-well plate in the same manner as in Example 1, using the polarized light source, a sample containing cells in each well from the upper side of the film covering the plate Was observed.
Even from the upper side of the film, as in Example 3, the bottom surfaces of all wells could be clearly imaged. Therefore, all the samples in the plate could be observed and analyzed at once. In addition, since the observation and analysis can be performed with the film attached, the liquid inside does not spill out, and the sterility inside the culture vessel can be maintained.

(Example 10)
A culture vessel having a lid-like member in which the COP film of Example 4 was adhered to the upper surface of a 96-well plate of Example 4 (manufactured by COP1060R) was created (the bottom and the lid are the same retardation member).
After performing sterilization treatment, cell culture and treatment using the 96-well plate in the same manner as in Example 2, using the polarized light source, a sample containing cells in each well from above the film covering the plate Was observed.
Even from the upper side of the film, as in Example 4, the bottom surfaces of all wells could be clearly imaged. Therefore, all the samples in the plate could be observed and analyzed at once. In addition, since the observation and analysis can be performed with the film attached, the liquid inside does not spill out, and the sterility inside the culture vessel can be maintained.

(Example 11)
A 96 well plate (manufactured by COP1060R) having the same phase difference as in Example 5 was prepared. Also, a cover (lid member) that covers the 96-well plate by injection molding was created so that the gap between the well and the cover was 0.2 mm so that it could be vented, and placed in an annealing furnace at 90 ° C. for 24 hours. A 96-well plate cover having a total phase difference of zero was prepared (the whole culture vessel was a zero phase difference member).
After performing sterilization treatment, cell culture, and treatment using the 96-well plate and cover in the same manner as in Example 5, the polarized light source was used to remove cells in each well from the upper side of the cover covering the plate. The containing sample was observed.
The bottom surfaces of all wells could be clearly imaged from the top of the cover. Therefore, it was possible to observe and analyze all the samples in the plate at the same time with the lid on. In addition, since the gap between the well and the cover is 0.2 mm, gas exchange can be performed with the cover on, so that the culture can be continued for a long period of time after observation and analysis.

(Example 12)
A plate-shaped member (material: COP1060R) having a thickness of 1 mm, a width of 15 mm, and a length of 60 mm is formed by injection molding, and two flow paths are formed on the surface of the plate-shaped member by machining to create a micro-channel chip. The COP film of Example 1 is pasted so as to cover the bottom surface of the channel, and the COP film of Example 1 is disposed on the surface of the microchannel chip so that the upper surface of the channel is covered with the film, A micro-channel chip having a shape-like lid was created (the channel and the lid were zero phase difference members).
After sterilization of the microchannel chip having a sheet-like lid by EOG, each channel was seeded with CHO cells at a concentration of 5 × 10 ⁵ cells / ml, and ham F12 medium supplemented with glucose was added. The culture was performed at 37 ° C. for 5 days under a carbon dioxide concentration of 5%. Then, using the polarized light source, the light that has passed through the polarizing plate is irradiated from the lower side to the microchannel chip, and from the upper side of the sheet-like lid, through each polarizing plate that can be rotated 360 ° C. Samples containing cells were observed. The photograph which imaged the microchannel chip | tip from the upper side of the sheet-like lid | cover is shown in FIG.
As shown in the photograph of FIG. 6, the bottom surfaces of both flow paths could be clearly imaged even from the upper side of the sheet-like lid. Therefore, all the samples in the microchannel chip could be observed and analyzed at a time. Moreover, since observation and analysis can be performed with the sheet-like lid attached, the liquid inside does not spill out and the sterility inside the culture vessel can be maintained.

(Example 13)
The COP film of Example 1 was bag-formed by heat welding so as to be a hexahedron having a side of 25 cm, and a culture bag was prepared (the whole culture vessel was a zero phase difference member).
After sterilizing the culture bag thus prepared with EOG, CHO cells were seeded in each channel at a concentration of 5 × 10 ⁵ cells / ml, and ham F12 medium supplemented with glucose was used for 7 days. The cells were cultured at 37 ° C. and a carbon dioxide concentration of 5%. Using the polarized light source, the sample containing the cells in the culture bag was observed through the polarizing plate that can be irradiated with light passing through the polarizing plate from the vertical direction and rotated at 360 ° C. from the vertical direction. . The photograph which imaged the bottom face of the culture bag from the upper direction of the culture bag is shown in FIG.
As shown in the photograph of FIG. 7, even from the outside of the culture bag, the bottom surface of the culture bag could be imaged uniformly and clearly without unevenness. Therefore, all the samples in the culture bag could be observed and analyzed at a time. Further, since observation and analysis can be performed without opening the culture bag, the liquid inside does not spill out, and the sterility inside the culture bag can be maintained.

  According to the culture container of the present invention, even when a plurality of samples are analyzed at the same time, observation and analysis can be performed with high accuracy without the influence of the material constituting the culture container.

Claims (7)

  A culture vessel at least partially formed of the same phase difference member or a zero phase difference member. A cylindrically shaped medium holding part having a bottom surface and a side wall,
Optionally comprising a lid-like member covering the upper surface of the culture medium holding unit,
The culture container according to claim 1, wherein at least two of the bottom surface, the side wall, and the lid-like member are formed of a zero phase difference member or the same phase difference member. It has two or more cylindrical medium holding parts having a bottom surface and a side wall,
Optionally provided with a lid-like member having two or more lid portions covering the upper surfaces of the two or more medium holding portions,
The culture container according to claim 1, wherein at least one of the two or more side walls, the two or more bottom surfaces, and the two or more lid parts is formed of a zero phase difference member or the same phase difference member. A plate-like member having a channel formed on the surface;
A lid-like member covering the flow path,
The culture vessel according to claim 1, wherein the flow path and the lid-like member are formed of a zero phase difference member or the same phase difference member. A plate-like member having two or more flow paths formed on the surface;
Optionally comprising a lid-like member having two or more lids covering each of the two or more flow paths;
The culture vessel according to claim 1, wherein at least two or more of the flow paths are formed of a zero phase difference member or the same phase difference member.   The culture container according to claim 4 or 5, wherein the lid member is a sheet member bonded to the plate member so as to cover the flow path.   The culture vessel according to any one of claims 1 to 6, wherein the zero phase difference member or the same phase difference member is made of a cycloolefin polymer.