JP7368470B2 - Method for manufacturing cell culture containers - Google Patents

Description

The present invention relates to a method for manufacturing a container for cell culture. More specifically, the present invention relates to a method for manufacturing a cell culture container, a defoaming method, and a method for manufacturing cells and spheroids using the cell culture container obtained by the method.

Conventionally, when cells are seeded into a culture container, the presence of air bubbles on the culture surface often results in poor culture, so degassing treatments such as ultrasonic degassing, vacuum degassing, and centrifugal degassing are performed before seeding. Cell culture is carried out by carrying out (for example, Patent Document 1).

JP2017-205021A

However, prior art methods are still unsatisfactory, such as requiring large-scale equipment or operating outside of a sterile environment, resulting in possible contamination. There is also a method of removing air bubbles by pre-wetting for a long time without performing the above-mentioned treatment, but since some residual air bubbles are observed and it takes time, further improved technology is desired. There is.

In addition, it has been newly discovered that there is a problem that air bubbles are difficult to escape from small-diameter culture compartments (wells).

An object of the present invention is to provide a method for manufacturing a novel container for cell culture, and a method for manufacturing cells and spheroids using the container for cell culture obtained by the method.

Another object of the present invention is to provide a simple and efficient method for manufacturing a cell culture container in which bubbles are less likely to exist.

A further object of the present invention is to provide a simple and efficient defoaming method for cell culture containers.

As a result of intensive studies to achieve the above object, the present inventors found that in culture vessels with small diameter culture compartments (wells), it is possible to easily transport the liquid to the compartments from a member having a specific diameter. They also discovered that the liquid can be efficiently filled with almost no bubbles, and after further study, they have completed the present invention.

That is, the present invention relates to the following [1] to [11].
[1] A method for manufacturing a cell culture substrate having a concave portion with an opening diameter of 1000 μm or less in diameter, and a cell culture container in which a culture solution is provided at least in the concave portion, the liquid feeding opening having a diameter of 3 mm or less A method for producing a cell culture container, the method comprising the step of feeding a liquid feeding liquid from the source and/or at a flow rate of 100 cm/s or more.
[2] A defoaming method for a cell culture substrate having a recess with an opening diameter of 1000 μm or less and a cell culture container in which a culture solution is provided at least in the recess, the liquid feeding port having a diameter of 3 mm or less A method for defoaming a cell culture container, in which a liquid is pumped from the bottom and/or at a flow rate of 100 cm/s or more.
[3] The method described in [1] or [2] above, wherein the liquid is delivered at a flow rate of 0.001 mL/s or more per 1 cm ² of opening area.
[4] The method according to any one of [1] to [3] above, wherein the liquid is fed from 0.1 to 10 mm above the recess.
[5] The method according to any one of [1] to [4] above, wherein the diameter of the liquid feeding port is 2.5 mm or less in diameter.
[6] The ratio of the diameter of the liquid feeding port to the diameter of the recess opening (diameter of the liquid feeding port/diameter of the recess opening) is 30 or less, according to any one of [1] to [5] above. the method of.
[7] The cell culture substrate has a plurality of recesses each having an opening diameter of 1000 μm or less in diameter, the inner surface of the recess has a cell non-adhesive surface, and the bottom surface of the recess has a cell adhesive property. The method according to any one of [1] to [6] above, which is a cell culture sheet having a surface.
[8] The method according to any one of [1] to [7] above, which is carried out in a sterile environment.
[9] The method according to any one of [1] to [8] above, wherein a liquid feeding liquid is injected into the cell culture substrate in advance before liquid feeding.
[10] A method for culturing cells using a cell culture container obtained by the method according to any one of [1] to [9] above.
[11] A method for producing spheroids, using a cell culture container obtained by the method according to any one of [1] to [9] above.

According to the present invention, a container for cell culture can be easily prepared, and the resulting container for cell culture is less likely to have air bubbles, making it possible to carry out cell culture efficiently.

FIG. 1 is micrographs before and after defoaming treatment in Example 1. FIG. 2 is a micrograph of spheroids formed in Example 1. FIG. 3 is micrographs before and after defoaming treatment in Example 2. FIG. 4 is micrographs before and after defoaming treatment in Example 3.

The method for manufacturing a cell culture container of the present invention includes feeding a liquid into a cell culture substrate having a recess with an opening diameter of 1000 μm or less from a liquid feeding port having a diameter of 3 mm or less and/or at a flow rate of 100 cm/ s or more, including the step of feeding the liquid for liquid feeding. Through this step, the cell culture container obtained includes the base material and the liquid in a state in which bubbles are not easily formed or left in the recesses. Therefore, in the cell culture container of the present invention, the amount of liquid in the entire container is not limited as long as at least the recesses are filled with liquid. The air bubbles in the recess may be visually confirmed. For example, when the culture solution is filled, the formation and remaining of air bubbles can be visually confirmed.

The production method of the present invention can be applied to any cell culture substrate that has a recess on its surface with an opening diameter of 1000 μm or less, and it is sufficient that cells can be cultured within the recess.

The diameter of the recess is not particularly limited as long as it is 1000 μm or less in diameter, and may be, for example, 800 μm or less, 600 μm or less, 500 μm or less, 400 μm or less, or 300 μm or less. Further, the lower limit is not particularly set, and may be, for example, 10 μm. Note that the diameter of the recess is the maximum diameter of the opening.

The area of the opening of the recess is, for example, within the range of 0.5 × 10 ^-3 to 5.0 × 10 -1 cm ² per recess, such as 1.0 × ^{10 -1 cm 2} and 5.0 × 10 ^-2 cm. ² , 2.0×10 ^-2 cm ² , 1.0×10 ^-2 cm ² , 5.0×10 ^-3 cm ² , 2.5×10 ^-3 cm ² , 2.0×10 ^-3 cm ² , 1.0×10 ^-3 cm ² etc. is exemplified.

The depth of the recess is not particularly limited as long as cells can be cultured, and can be appropriately set according to the common general knowledge in the art depending on the size of the cells to be cultured, the method of forming the recess, and the like. For example, the depth of the recess may be 10 nm or more (for example, 100 nm or more), etc., or within the range of 1 μm or more [for example, 5 μm or more, 10 μm or more (for example, 10 to 1000 μm, 10 to 300 μm)], etc. There may be.

The shape of the opening and the bottom (bottom) of the recess is not limited to a circle, and may be, for example, a polygon or an ellipse, and the shape of the bottom may be the same as or different from the shape of the opening. good. Further, the bottom surface may be flat, curved, or smooth, but it is more desirable that the bottom surface be flat and/or smooth. The area of the bottom surface is, for example, within the range of 0.5×10 ^-3 to 5.0×10 ^-1 cm ² per recess, such as 1.0×10 ^-1 cm ² , 5.0×10 ^-2 cm ² , 2.0 ×10 ^-2 cm ² , 1.0 × 10 ^-2 cm ² , 5.0 × 10 ^-3 cm 2 , 2.5 × 10 ^-3 cm ² , 2.0 × 10 ^-3 cm ² , 1.0 × 10 ^-3 cm ² etc. are exemplified ^. Ru. In addition, the diameter (length) of the bottom surface may be the same as or different from the diameter of the opening, and even if the diameter of the bottom surface is smaller than the diameter of the opening, the diameter of the bottom surface may be the same as the diameter of the opening. It may be larger than the caliber of the The diameter of the bottom surface is, for example, in the range of 10 to 1000 μm, 10 to 700 μm, 10 to 600 μm, 10 to 500 μm, 10 to 400 μm, and 10 to 300 μm. For example, when the diameter of the bottom surface is the same as that of the opening, the shape of the recess is a columnar shape, and especially when the shapes of the opening and the bottom surface are circular, a columnar shape is formed. When the diameter of the bottom surface is smaller than the diameter of the opening, the shape of the recess is tapered toward the bottom surface of the recess.

The ratio of the bottom pore diameter to the opening pore diameter (bottom pore diameter/opening pore diameter) is not particularly limited, but from the viewpoint of ease of cell seeding, etc., it is 5/1 to 1/5. , 3/1 to 1/3, and 1/1 to 1/2 are exemplified.

In addition, the distance (gap) between the opening and the adjacent opening is not particularly limited, but may be, for example, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, depending on the desired mass culture. Hereinafter, ranges such as 300 μm or less, 200 μm or less, and 100 μm or less are exemplified, and any finite value may be used.

The ratio of the opening pore diameter to the recess depth (opening pore diameter/recess depth) is not particularly limited, but from the viewpoint of ease of cell seeding, etc., it is 5/1 to 1. Examples include /5, 3/1 to 1/3, and 2/1 to 1/1. When it is within the above range, cells are less likely to jump out of the recesses, and may be advantageous in terms of defoaming.

The number of recesses cannot be set unconditionally depending on the area of the substrate, the type of cells to be cultured, etc., but can be set as appropriate according to the common general knowledge in the art. For example, the lower limit number per unit area (cm ² ) can be 1, 10, 30, 50, etc., and the upper limit can be 1000, 500, 300, 200, 100, etc. . Further, the total number of recesses on the surface of the base material can be set as appropriate, for example, 10 or more, 100 or more, 1000 or more, 10000 or more, 50000 or more.

The material of the cell culture substrate in the present invention is not particularly limited as long as it has the above-mentioned recesses on its surface. According to the study of the present invention, the higher the hydrophobicity of the material for the cell culture substrate, the stronger the adhesion of air bubbles. Air bubbles can be removed. Examples of highly hydrophobic materials include fluororesins, styrene resins (such as polystyrene), polyolefin resins, and polyimide resins, but are not particularly limited and may be any known material. Furthermore, the entire cell culture substrate may be made of the same material or may be made of a plurality of materials. Specifically, for example, the recess and the surface of the base material may be made of different materials, and furthermore, the material may be made of different materials depending on the position within the recess.
For example, as an example of a cell culture substrate in which the inner surface of the recess and the bottom surface of the recess are made of different materials, the inner surface of the recess has a cell non-adhesive surface, and the bottom surface of the recess has a cell adhesive surface. Examples include cell culture sheets having the following. Such a cell culture sheet has the above-mentioned surface characteristics in addition to the small diameter of the opening, so that, for example, when a culture medium is simply injected, air bubbles remain in the recesses as shown in FIG. 1 (non-defoamed). However, the present invention makes it difficult to visually confirm the formation or remaining of air bubbles in the recesses.

A "cell-adhesive surface" is a surface to which, for example, when cells settle on the surface in a solution used for culture, the cells will adhere to the surface at certain attachment points. . The cell-adhesive surface only needs to be made of a substance that exhibits at least cell-adhesive properties.

The substance exhibiting cell adhesive properties is not particularly limited and can be used as long as it can bind to cell surface molecules such as proteins and sugar chains present in the cell membrane of the cells used. It may be either hydrophilic or hydrophobic, but from the viewpoint of cell adhesion, spheroid formation, etc., hydrophilic (especially hydrophilic but not superhydrophilic) or hydrophobic (In particular, those that are hydrophobic but not superhydrophobic) are preferred, and those that exhibit hydrophobicity are even more preferred. Further, the degree of adhesiveness of the substance exhibiting cell adhesiveness may be such that cells do not jump out from within the recess.
Examples of such substances include substances obtained from living organisms or synthesized, such as proteins (collagen, fibronectin, laminin, etc.) and synthetic resins (styrene resins, polyolefin resins, etc.). , polycycloolefin resins, polyethylene terephthalate resins, acrylic resins, fluororesins, polyimide resins, polysulfone resins, polyethersulfone resins, polydimethylsiloxane resins, mixtures and surface-modified products thereof, etc.). When a synthetic resin is selected, a cell culture sheet with excellent handling properties can be obtained due to the strength and heat resistance of the synthetic resin itself. In addition, from the viewpoint of biocompatibility, from the viewpoint of improving the uniformity of cultured cells or spheroids obtained using the sheet, or from the viewpoint of facilitating medium exchange work by appropriately adhering to various cells. It is preferable to select synthetic resins such as , styrene resins, polyolefin resins, and polyimide resins. By selecting non-biological components such as styrene resin, polyolefin resin, and polyimide resin, spheroids obtained using cell culture substrates containing styrene resin, polyolefin resin, and polyimide resin can be used for regenerative medicine. This makes it easy to apply to fields such as drug discovery and drug discovery.

Polyimide resin can be suitably used for cell culture as described above, but it is usually hydrophobic and when applied to a substrate with small diameter recesses as described above, it is difficult to simply inject the liquid into it. If it is used alone, bubbles will be formed in the recesses and it will be difficult to remove them, so it is likely to be avoided as a material for the base material. However, according to the present invention, it is possible to apply the present invention to a base material having a concave portion having the diameter as described above. Examples of polyimide resins include polyimide resins containing structural units represented by the following formula (I). Further, from the viewpoint of good spheroid formation, a resin having a fluorine atom in the molecule is preferable, and a fluorine-containing polyimide (fluorine-containing polyimide resin) is more preferable. The polyimide resin used in the present invention is typically obtained by imidizing a polyamic acid obtained by polymerizing one or more acid dianhydrides and diamines. The polyimide resin may include polyamic acid as part of its chemical structure. The polyimide resin may be produced by any known method. As an example, a two-step synthesis method can be used. The two-step synthesis method for polyimide resin is a method in which polyamic acid is synthesized as a precursor and the polyamic acid is converted into polyimide. The polyamic acid as a precursor may be a polyamic acid derivative. Examples of the polyamic acid derivatives include polyamic acid salts, polyamic acid alkyl esters, polyamic acid amide, polyamic acid derivatives from bismethylidene pyromellitide, polyamic acid silyl esters, polyamic acid isoimides, and the like. Polyimides include acid anhydrides such as pyromellitic dianhydride, biphenyltetracarboxylic dianhydride, and benzophenonetetracarboxylic dianhydride, and diamines such as oxydiamine, paraphenylenediamine, metaphenylenediamine, and benzophenonediamine. An example is polyimide. Examples of the resin having a fluorine atom include 4,4'-hexafluoroisopropylidene diphthalic anhydride (6FDA)/1,4-bis(aminophenoxy)benzene (TPEQ) copolymer, 6FDA/1,3 -Bis(4-aminophenoxy)benzene (TPER) copolymer, 6FDA/4,4'-oxydiphthalic anhydride (ODPA)/TPEQ copolymer, 4,4'-(4,4'-isopropylidene phenoxy)diphthalic acid (BPADA)/2,2-bis[4-(4-aminophenoxy)phenyl]hexafluoropropane (HFBAPP), 6FDA/2,2-bis(4-(4-aminophenoxy)phenyl)propane Examples include fluorine-containing polyimide resins containing structural units represented by the following formula (I) such as (BAPP) copolymers; ethylene-tetrafluoroethylene copolymers, and the like.

In the above formula (I), X ⁰ represents any of an oxygen atom, a sulfur atom, a carbonyl group, a sulfonyl group, or a divalent organic group;
Y represents a divalent organic group;
Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ and Z ⁶ each independently represent a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom,
p is 0 or 1.
In addition, in the polyimide resin, the chemical structure represented by formula (I) may be different for each constituent unit of the resin, or may be the same. It is preferable that at least one of X ⁰ , Y, Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , and Z ⁶ contains one or more fluorine atoms.

In the above formula (I), when p=0, X ⁰ may not exist (in other words, the left and right benzene rings may be directly bonded), but when p=1 , the left and right benzene rings are bonded via X ⁰ .

Specific examples of the divalent organic group represented by X ⁰ include an alkylene group, an arylene group, an aryleneoxy group, an arylenethio group, and the like. Further, it may be a fused cyclic divalent hydrocarbon group, a heterocyclic fused cyclic divalent hydrocarbon group, or an oxy group or thio group thereof. Among these, an alkylene group, an aryleneoxy group, and an arylenethio group are preferred, and an alkylene group and an aryleneoxy group are more preferred, and these may be substituted with a fluorine atom. The alkylene group has, for example, 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms.

Examples of the alkylene group substituted with a fluorine atom, which is an example of X ⁰ , include -C(CF ₃ ) ₂ -, -C(CF ₃ ) ₂ -C(CF ₃ ) ₂ -, etc. . Among the alkylene groups mentioned above as examples of X ⁰ , -C(CF ₃ ) ₂ - is preferred.

Examples of the arylene group that is an example of X ⁰ include the following.

Examples of the aryleneoxy group that is an example of X ⁰ include the following.

Examples of the arylenthio group that is an example of X ⁰ include the following.

From the viewpoint of forming spheroids well on the substrate, the divalent organic group represented by X ⁰ is selected from the group consisting of b-2 to b-10 and c-2 to c-10 above. It may be a structure selected from the group consisting of b-7 to b-9 and c-7 to c-9, or a structure represented by b-8.

The ^above -mentioned arylene group, aryleneoxy group, and arylenethio group, which are examples of (more preferably a fluorine atom), a methyl group, and a trifluoromethyl group. There may be a plurality of these substituents, and in that case, the types of the substituents may be the same or different. Suitable substituents for the arylene group, aryleneoxy group and arylenethio group are a fluorine atom and/or a trifluoromethyl group, preferably a fluorine atom. The arylene group, aryleneoxy group, and arylenethio group are preferably substituted with at least one fluorine atom when Y does not contain a fluorine atom.

In the above formula (I), the divalent organic group represented by Y is not particularly limited, but includes, for example, a divalent organic group having an aromatic ring. Specifically, a group consisting of one benzene ring, or a group having a structure in which two or more benzene rings are bonded via a carbon atom (i.e., a single bond or an alkylene group), an oxygen atom, a sulfur atom, or directly. Can be mentioned. Specifically, the following groups can be exemplified.

The above-mentioned divalent organic group having an aromatic ring, which is an example of Y, is a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, preferably a fluorine atom or a chlorine atom), if substitutable. , more preferably a fluorine atom), a methyl group, and a trifluoromethyl group. There may be a plurality of these substituents, and in that case, the types of the substituents may be the same or different. A preferred substituent for a divalent organic group having an aromatic ring is preferably a fluorine atom and/or a trifluoromethyl group, particularly when X ⁰ does not contain a fluorine atom, and more preferably is a fluorine atom.

From the viewpoint of spheroid formation, in the above formula (I), Y is a structure selected from the group consisting of d-3, d-9, e-1 to e-4, f-6, and f-7. The structure is preferably e-1, e-3 or e-4.

In the above formula (I), Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , and Z ⁶ may be the same or different, and each independently represents a hydrogen atom or a fluorine atom. , a chlorine atom, a bromine atom, or an iodine atom, and when at least one of X ⁰ and Y does not contain a fluorine atom, at least one of Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , and Z ⁶ It is preferable that one is a fluorine atom.

From the viewpoint of spheroid formation, in a preferred embodiment of the present invention, in the above formula (I), the divalent organic group represented by X ⁰ is -C(CF ₃ ) ₂ -, the above b-2 to b -10 and c-2 to c-10; and Y is selected from the group consisting of d-3, d-9, e-1 to e-4, f-6, and f-7. selected. In a more preferred embodiment of the present invention, in the above formula (I), the divalent organic group represented by X ⁰ is -C(CF ₃ ) ₂ -, b-7 to b-9 and c-7 to and Y is selected from the group consisting of e-1, e-3 and e-4.

The polyimide resin consisting of the structural unit represented by the above formula (I) can be obtained by firing a polyamic acid obtained by polymerizing an acid dianhydride and a diamine. Note that the imidization rate of the above-mentioned "polyimide resin consisting of the structural unit represented by formula (I)" may not be 100%. That is, the polyimide resin composed of the structural unit represented by the formula (I) may be composed only of the structural unit represented by the above formula (I), but as long as the objective effect of the present invention is not impaired. , a constituent unit in which the cyclic imide structure remains an amic acid without undergoing dehydration and ring closure may be included in part.

The polyamic acid synthesis reaction is preferably carried out in an organic solvent. The organic solvent used in the polyamic acid synthesis reaction is not particularly limited as long as it can efficiently react with the raw material acid dianhydride and diamine and is inert to these raw materials. . For example, N-methylpyrrolidone (NMP), N,N-dimethylacetamide, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, sulfolane, methylisobutylketone, acetonitrile, benzonitrile, nitrobenzene, nitromethane, acetone, methylethylketone, isobutylketone , polar solvents such as methanol; non-polar solvents such as toluene and xylene. Among these, it is preferable to use a polar solvent. These organic solvents may be used alone or as a mixture of two or more. The reaction mixture after the amidation reaction may be directly subjected to thermal imidization. The concentration of the polyamic acid in the polyamic acid solution is not particularly limited, but from the viewpoint of the polymerization reactivity of the resulting resin composition, the viscosity after polymerization, and the ease of handling during subsequent film formation and baking, it is preferably , 5% by weight or more, more preferably 10% by weight or more, preferably 50% by weight or less, more preferably 40% by weight or less. The viscosity of the resin composition is not particularly limited, but can be measured according to a known measuring method, and is, for example, within the range of 1 to 20 Pa·s, preferably 3 to 15 Pa·s at 23°C. .

The polyamic acid is imidized by either thermal imidization or chemical imidization to obtain a resin composition containing a fluorine-containing polyimide. In a specific embodiment, the polyamic acid is imidized by heat treatment (thermal imidization) to obtain a resin containing fluorine-containing polyimide. Polyimide obtained by thermal imidization has no possibility of residual catalyst and is more preferable for cell culture applications.

When imidizing by thermal imidization, for example, the polyamic acid is heated in air, or more preferably in an inert gas atmosphere such as nitrogen, helium, or argon, or in vacuum, preferably at a temperature of 50 to 400°C. A resin composition containing polyimide can be obtained by performing an imidization reaction by firing under conditions of, more preferably 100 to 380°C, preferably 0.1 to 10 hours, more preferably 0.2 to 5 hours.

The polyamic acid to be subjected to the thermal imidization reaction is preferably dissolved in an appropriate solvent. The solvent may be any solvent as long as it dissolves the polyamic acid, and the solvents mentioned above regarding the polyamic acid synthesis reaction can also be used.

In the case of imidization by chemical imidization, the polyamic acid can be directly imidized by using a dehydration cyclization reagent described below in a suitable solvent.

The dehydration cyclization reagent can be used without particular limitation as long as it has the effect of chemically dehydrating and cyclizing polyamic acid to form polyimide. As such a dehydration cyclization reagent, imidization can be efficiently promoted by using a tertiary amine compound alone or in combination with a tertiary amine compound and a carboxylic acid anhydride. It is preferable.

Examples of the tertiary amine compound include trimethylamine, triethylamine, tripropylamine, tributylamine, pyridine, 1,4-diazabicyclo[2.2.2]octane (DABCO), 1,8-diazabicyclo[5.4. 0] undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, N,N,N',N'-tetramethyldiaminomethane, N,N,N',N' -tetramethylethylenediamine, N,N,N',N'-tetramethyl-1,3-propanediamine, N,N,N',N'-tetramethyl-1,4-phenylenediamine, N,N,N ',N'-tetramethyl-1,6-hexanediamine, N,N,N',N'-tetraethylmethylenediamine, N,N,N',N'-tetraethylethylenediamine, and the like. Among these, pyridine, DABCO, and N,N,N',N'-tetramethyldiaminomethane are particularly preferred, and DABCO is more preferred. The number of tertiary amines may be one, or two or more.

Examples of the carboxylic anhydride include acetic anhydride, trifluoroacetic anhydride, propionic anhydride, butyric anhydride, isobutyric anhydride, succinic anhydride, maleic anhydride, and the like. Among these, acetic anhydride and trifluoroacetic anhydride are particularly preferred, and acetic anhydride is more preferred. The number of carboxylic acid anhydrides may be one, or two or more.

As a solvent for dissolving polyamic acid in chemical imidization, a polar solvent with excellent solubility is suitable. Examples include tetrahydrofuran, N,N-dimethylacetamide, N,N-dimethylformamide, N-methylpyrrolidone, dimethylsulfoxide, etc. Among these, N,N-dimethylacetamide, N,N-dimethylformamide and N -Methylpyrrolidone is preferred from the viewpoint of homogeneous reaction. When these solvents are used as a solvent for the amidation reaction, the polyamic acid can be directly used for chemical imidization without being separated from the reaction mixture after the amidation reaction.

The weight average molecular weight of the polyimide resin is, for example, 5,000 to 2,000,000, preferably 8,000 to 1,000,000, and more preferably 20,000 to 500,000. In addition, in this specification, the weight average molecular weight of the resin can be measured according to a known measuring method, and by having a weight average molecular weight within the above range, the synthesis and handling, film formation, and spheroid forming properties of the polyimide resin are improved. Becomes good.

The cell-adhesive surface may further contain additive components such as plasticizers and antioxidants in addition to the above-mentioned substances exhibiting cell-adhesive properties.

Substances exhibiting cell adhesive properties or cell adhesive surfaces [hydrophobic (especially hydrophobic but not superhydrophobic) substances exhibiting cell adhesive properties or cell adhesive surfaces] have a static water contact angle of 70° or more. The falling angle may be 15° or more, or the static water contact angle may be 70° or more and the falling angle may be 15° or more.
In particular, substances exhibiting cell adhesive properties or cell adhesive surfaces [hydrophobic (especially hydrophobic, not superhydrophobic) substances exhibiting cell adhesive properties or cell adhesive surfaces] have a static water contact angle of 70° or more. It is preferable that When the cell adhesive surface satisfies these conditions, spheroid formation is further promoted. From the viewpoint of spheroid formation, the static water contact angle is more preferably more than 75°, even more preferably 77° or more, even more preferably 79° or more, particularly preferably 80° or more (for example, more than 80°). and the upper limit of the static water contact angle is, for example, less than 150°, preferably 120° or less (e.g. less than 120°), more preferably 110° or less, especially 100° or less (e.g. 99 98° or less, 97° or less, 95° or less), and more preferably less than 90°.

On the other hand, the static water contact angle of a substance exhibiting cell adhesiveness or a cell adhesive surface [a hydrophilic (especially hydrophilic but not superhydrophilic) substance exhibiting cell adhesiveness or a cell adhesive surface] is preferably 65 The angle may be less than or equal to 55°, more preferably less than or equal to 55°, and even more preferably less than or equal to 50°. Note that the lower limit may be 0° or more, preferably 5° or more, and more preferably 10° or more.
From the same point of view, the falling angle of a substance exhibiting cell adhesive properties or a cell adhesive surface is 18° or more, 19° or more, 20° or more, 22° or more, 24° or more, 26° or more, 28° or more. , 30° or more, and the higher the angle, the better. The upper limit of the falling angle is, for example, less than 80°, preferably 70° or less (for example, less than 70°), more preferably 60° or less (for example, less than 60°), and still more preferably 50° or less ( e.g. less than 50°).

Note that such static water contact angle and falling angle may be values on a cell-adhesive surface, or values on a substance exhibiting cell-adhesiveness (or a substance constituting a cell-adhesive surface). good.
Note that the surface characteristics can be measured according to a known method. For example, the static water contact angle and falling angle may be measured, for example, by the method described below.

When the surface exhibiting cell adhesiveness constitutes the bottom surface of the recess, the proportion of the cell adhesive substance occupying the bottom surface of the recess is not particularly limited, but is 90% or more, 95% of the bottom surface of the recess. It is preferable that it occupies 99% or more, or substantially the entire bottom surface. Further, the cell-adhesive surface on the bottom is preferably flat (flat-bottomed, plate-like) from the viewpoint of observability.

"Cell non-adhesive surface" means, for example, that when cells settle on the surface in a solution used for culture, the cells hardly change their shape, do not adhere at all, or temporarily adhere to the surface. A surface that naturally detaches even if it is weakly bonded. The cell non-adhesive surface only needs to be made of a substance that exhibits at least cell non-adhesive properties.

Substances exhibiting cell non-adhesion properties are not particularly limited and can be used as long as the cells used for culture do not adhere to them or do not bind to cell surface molecules such as proteins and sugar chains present in the cell membrane of the cells used. It may or may not be biocompatible.

Furthermore, the substance exhibiting cell non-adhesiveness (or cell non-adhesive surface) may be either hydrophobic or hydrophilic; for example, it may be superhydrophobic (superhydrophobic). It may be a superhydrophilic material or a superhydrophilic material. From the viewpoint of cell non-adhesiveness, spheroid uniformity, formation property, etc., hydrophobic (especially superhydrophobic) [e.g., hydrophobic or hydrophilic (especially hydrophobic) cell adhesive surfaces or Substances that are more hydrophobic (especially superhydrophobic) with respect to the resin (and its contact angle) are particularly preferred; ) or a substance that is more hydrophilic (especially superhydrophilic) with respect to the resin constituting the surface (and its contact angle) is also preferable.
Examples of such substances include (poly)ethylene glycol and its derivatives, MPC (2-methacryloyloxyethylphosphorylcholine) and its derivatives, HEMA (hydroxyethyl methacrylate) and its derivatives, etc. Compounds containing or polymers of these compounds [e.g., poly-HEMA (polyhydroxyethyl methacrylate), etc.], SPC (segmented polyurethane) and its derivatives, proteins obtained from living organisms (albumin, etc.), and cells. Non-adhesive sugar chains (agarose, cellulose, etc.) can be appropriately selected and used depending on the type of cell. Among these, from the viewpoint of adhesion with cell adhesive surfaces and synthetic resins, from the viewpoint of simplifying the manufacturing process of cell culture substrates or cell culture sheets, and from the viewpoint of uniformity of cultured cells or spheroids obtained. From the viewpoint of improved properties, MPC, derivatives thereof, or polymers thereof are preferred.

The substance exhibiting cell non-adhesion properties may be appropriately modified depending on ease of handling, desired degree of hydrophobicity (e.g., superhydrophobicity)/hydrophilicity (e.g., superhydrophilicity), etc. It's okay. For example, hydrophilicity and low solubility in water may be achieved by crosslinking a hydrophilic substance. In addition, the raw material (e.g., hydrophobic or hydrophilic) may be subjected to appropriate hydrophobic treatment or hydrophilic treatment (e.g., introduction of a hydrophobic group or hydrophilic group, etc.) to obtain the desired hydrophobic or hydrophilic property. You may obtain the material.

Immobilization of substances that exhibit cell non-adhesion can be achieved by drying a solution containing these on the surface of the substrate, by melting the substance and pressing it, or by applying energy rays such as UV to the substance applied to the substrate. Covalent bonds are formed by causing a chemical reaction (for example, a condensation reaction between functional groups such as carboxyl groups and amino groups) between the functional groups of the substance and the functional groups on the base material. The substance can be immobilized on the surface of the substrate by a method of bonding a thiol group of the substance to a thin film of metal (platinum, gold, etc.) formed in advance on the substrate. The thickness upon immobilization is not particularly limited, and is exemplified by 0.01 to 1000 μm.

When the surface exhibiting cell non-adhesion constitutes the inner surface of the recess, the proportion of the surface exhibiting cell non-adhesion occupying the inner surface of the recess is not particularly limited, but is to the extent that the adhesiveness of cultured cells is reduced. It is preferable that Preferably, it occupies 90% or more of the area of the inner surface, more preferably 95% or more, particularly preferably all of it.
A surface that exhibits cell non-adhesion can be determined using, for example, the static water contact angle as an indicator of its surface characteristics, from the perspective of making the size of cultured cells uniform and improving circularity. . For example, in the case of a hydrophobic surface made of a substance exhibiting cell non-adhesion, the static water contact angle is preferably 90° or more, more preferably 93° or more, and still more preferably 95° or more. Further, the angle may be 150° or less, preferably 130° or less, and more preferably 120° or less.
On the other hand, in the case of a hydrophilic surface, the static water contact angle is preferably 65° or less, more preferably 55° or less, even more preferably 50° or less. Further, the angle may be 0° or more, preferably 5° or more, and more preferably 10° or more.
To give an example of such a material, a highly hydrophobic MPC (or a surface formed with the MPC) has a static water contact angle of, for example, 90° or more, 100° or more. can be realized.

Note that such a static water contact angle may be a value on a cell non-adhesive surface, or a value on a substance exhibiting cell non-adhesion (or a substance constituting a cell non-adhesive surface). good.
Further, the static water contact angle may be measured, for example, by the method described below.

From the viewpoint of improving the size uniformity and circularity of the obtained spheroids, it is preferable to balance the degree of adhesion between the cell adhesive surface and the cell non-adhesive surface. Therefore, even if the non-cell-adhesive surface exhibits adhesion to cells, it only needs to have lower adhesion than the cell-adhesive surface. For example, when the above static water contact angle is used as an index, the difference in static water contact angle (or its absolute value) on a cell non-adhesive surface (or a substance exhibiting cell non-adhesive property) is 3° or more (e.g. , 5° or more), more preferably 10° or more (for example, 12° or more), even more preferably 15° or more. In addition, the upper limit of the above difference (or its absolute value) is based on the hydrophobicity/hydrophilicity of the cell non-adhesive surface (or the substance showing cell non-adhesion) and the cell adhesive surface (or the substance showing cell adhesion). It can be selected as appropriate depending on the combination, etc., and may be, for example, 100°, 90°, 80°, 70°, 60°, 50°, 40°, 30°, etc., although it is not particularly limited.

A surface having these characteristics is obtained by physically or physically applying a substance having the characteristics to the surface of the base material so that the inner surface of the recess becomes a cell non-adhesive surface and the bottom surface of the recess becomes a cell adhesive surface. It may be formed by being chemically fixed or arranged, or the corresponding portion may be formed of a sheet-like material made of a substance having the relevant characteristics.

Further, in the cell culture sheet described above, the sheet surface, which is also the edge portion of the recess, may have a cell non-adhesive surface from the viewpoint of simplifying the production of the cell culture sheet. The cell non-adhesive surface of the sheet surface may be the same cell non-adhesive surface as the inner surface of the recess, or may be a different cell non-adhesive surface. In the case of the same cell non-adhesive surface, the sheet surface and the inner surface of the recess may have a continuous surface.

A cell culture sheet having recesses having such a configuration may be a layered structure including a layer including the bottom surface of the recess and a layer including the inner surface of the recess. Here, the layer that includes the inner surface of the recess is a layer that itself has a through hole. Therefore, one embodiment of the sheet is a laminate including a layer having a cell non-adhesive surface having through-holes and a layer having a cell adhesive surface.

A layer having a cell non-adhesive surface with through holes is made by fixing a substance exhibiting cell non-adhesive property to a layered base material from the viewpoint of forming through holes or simplifying the production of cell culture sheets. Preferably.

As the layered base material, any material known in the technical field can be used. For example, synthesis of polystyrene, polyethylene, polypropylene, polycarbonate, polyamide, polyacetal, polyester (polyethylene terephthalate, etc.), polyurethane, polysulfone, polyacrylate, polymethacrylate, polycycloolefin, polyetherketone, polyetheretherketone, polyimide, silicone, etc. Examples include plate-shaped bodies made of resin, synthetic rubber such as EPDM (ethylene propylene diene rubber), natural rubber, glass, ceramic, metal materials such as stainless steel, and the like. A transparent base material is also one of the preferable forms.

From the viewpoint of workability, it is preferable to immobilize a substance exhibiting cell non-adhesion to a layered base material after forming through-holes, which will be described later.

Preferably, the wall of the through hole corresponds to the inner surface of the recess described above, and the hole diameter and shape of the opening and the opposite end can be set in the same manner as the recess described above. Further, the depth of the through hole corresponds to the thickness of the layer having the cell non-adhesive surface, but it also corresponds to the depth of the above-mentioned recess, and can be set in the same manner as the recess. In addition, when a substance exhibiting cell non-adhesiveness is immobilized on a layered base material, the layer of the substance exhibiting cell non-adhesiveness preferably has a thickness of, for example, 1 nm or more, more preferably 10 nm or more. The thickness of the entire layer including the cell non-adhesive substance layer and the base material can be set as appropriate as long as it falls within the thickness range of the layer having the cell non-adhesive surface described above.

The formation of the through-hole is not particularly limited as long as the through-hole of the above-mentioned size can be formed, and any method can be performed. For example, it can be formed by drilling (drill, etc.), optical micromachining (laser (eg, CO ² laser, excimer laser, semiconductor laser, YAG laser), etc.), etching, embossing, etc. In the processing, the shape of the through hole may be tapered, and in this case, the periphery of the end portion is deformed, and the peripheral portion of the opening and the opening adjacent to the opening are deformed. A structure may be formed in which the layer thicknesses of the portions located in the intermediate region are different.

The thickness of the layer having a cell adhesive surface is, for example, 1 nm or more and 4 mm or less, preferably 1 μm or more and 1 mm or less, and may be the same as the bottom thickness of the recess.

The cell culture sheet may further include an adhesive layer (adhesive layer) between the layer having the cell adhesive surface and the layer having the cell non-adhesive surface.

As the adhesive layer, any adhesive layer known in the technical field can be used. Examples include acrylic resins, silicone resins, synthetic rubbers, natural rubbers, etc., and preferably a low elution adhesive layer can be used. Commercially available double-sided tape or the like may also be used.

The thickness of the adhesive layer is not particularly limited, and can be appropriately set within a range that does not impair the effects of the present invention. For example, 0.5 to 100 μm is exemplified.

The cell culture sheet can be manufactured by laminating in this order a layer having a cell non-adhesive surface having through holes and a layer having a cell adhesive surface. Layers other than the above may be laminated, and layers having cavities may be laminated.

When laminating each layer, each layer prepared in advance may be laminated in order, a layer may be formed separately on a layer prepared in advance, or a combination of these may be used. Good too. Specifically, for example, a substance exhibiting cell adhesion is cast, spray coated, or A layer having a cell-adhesive surface can be formed into a sheet by coating to an appropriate thickness and heating using methods such as dip coating, spin coating, and roll coating. On the other hand, a layer having a cell non-adhesive surface is prepared in advance by forming through holes in the base material of the layer having a cell non-adhesive surface and coating the surface with a substance exhibiting cell non-adhesive property. I can do it. Then, after peeling off the release sheet of the layer having a cell-adhesive surface molded above, it can be manufactured by laminating a separately prepared layer having a cell-non-adhesive surface. In addition, in laminating a layer with a cell-nonadhesive surface and a layer with a cell-adhesive surface, the above-mentioned adhesive layer (adhesive layer) may be used for lamination, or welding (high-frequency welding, ultrasonic welding) may be used. etc.), or may be laminated by compression bonding (thermocompression bonding, etc.).

The thickness of the cell culture sheet is not particularly limited, but from the viewpoint of ease of handling, it is preferably 10 to 5000 μm, more preferably 10 to 2000 μm. The sheet area is also not particularly limited, and is exemplified by, for example, 0.01 to 10,000 cm ² , preferably 0.03 to 5,000 cm ² .

The thus obtained cell culture substrate such as a cell culture sheet may be appropriately sized according to the size of the target device from the viewpoint of being used as is by being installed in a known cell culture device. Cell culture can be carried out by placing a medium containing cells on one surface of the substrate, and the cell culture substrate can be placed on a culture plate, each well of the plate, a culture dish (culture dish), a flask, a culture bag, etc. Cell culture can be carried out on a part or the entire surface of the cell culture substrate which is housed and fixed in a housing and fixed.

A liquid is sent to a container containing such a cell culture substrate. The device or instrument used for liquid feeding is, for example, one having at least a liquid feeding port having a diameter of 3 mm or less. The liquid feeding port may have a liquid feeding port with a diameter of 3 mm or less, for example, it may have a diameter of 2.5 mm or less, 2.0 mm or less, or 1.7 mm or less. In addition, from the viewpoint of liquid feeding flow rate and liquid feeding flow rate for removing air bubbles from the recess, the ratio of the diameter of the liquid feeding port to the diameter of the recess opening (diameter of the liquid feeding port/diameter of the recess opening) is It is preferably 30 or less, more preferably 20 or less, even more preferably 10 or less. The lower limit is not particularly limited, and can be, for example, 0.1 or more. Note that the diameter of the liquid feeding port means the maximum inner diameter of the liquid feeding port.

The shape and material of the liquid feeding port are not particularly limited as long as it has the diameter described above. For example, it is possible to use a known dispenser manufactured or adjusted to have the above-mentioned diameter at the liquid feeding port, or a syringe needle, blunt cannula, tip, sonde, etc. having the above-mentioned diameter.

Moreover, it is preferable that the liquid feeding device or instrument has a member for holding the liquid feeding liquid in addition to the liquid feeding port. Specifically, a cartridge, tank, or bottle may be used, and a syringe or the like may also be used. Its size (capacity), shape, and material are not particularly limited. The member may be coupled indirectly or directly to the liquid feeding port via a tube or the like, or may be formed integrally.

The liquid for liquid delivery is not particularly limited. The culture solution itself may be used, or physiological saline, buffer solution, etc. may be used. If a culture medium is not used as the liquid for feeding, remove the liquid filled in the recess after sending the liquid, and at that time, remove it until it is not completely gone, and then replace it with the culture medium. By this, or by directly adding the culture solution after sending the solution, the concave portions of the cell culture substrate can be filled with the culture solution. In addition, before the liquid feeding, the liquid feeding liquid can be injected into the cell culture substrate in advance and then the liquid feeding can be carried out, and when this pre-injection is performed, air bubbles are confirmed in the recess. Good too. There are no particular limitations on the method of pre-injection or the amount of liquid for liquid delivery.

The liquid feeding method is not particularly limited as long as the liquid can flow out from the liquid feeding port and fill the inside of the recess with the liquid feeding liquid. Specifically, for example, there is a method in which the tip of the liquid feeding port is placed 0.1 to 10 mm above the upper end surface of the opening of the recess, and the liquid is fed. At this time, the tip of the liquid feeding port may be placed at a position where it is immersed in a liquid that has been injected in advance into the culture container in which the substrate is installed. The number of liquid feeding ports to be arranged may be one or two or more per base material. Further, the liquid may be fed to a plurality of recesses at once, or the cell culture substrate itself or the liquid feeding port may be moved to feed the liquid continuously or intermittently. Moreover, it may be automated or manual. After the liquid is once delivered, the liquid present above the recess may be sucked, and the liquid may be sent again above the same recess or above a different recess. In this case, repeating small amounts of liquid feeding and suction tends to cause bubbles, so the amount of liquid fed at one time may be, for example, 10 mL or more, and is exemplified by 10 to 50 mL.

The liquid feeding flow rate can be, for example, 100 cm/s or more. Further, the speed may be 200 cm/s or more, 250 cm/s or more, 300 cm/s or more, etc. Further, the upper limit includes a flow rate at which the cell non-adhesive substance on the base material does not peel off or the base material is not damaged, such as 2000 cm/s.

Further, the liquid feeding flow rate is not particularly limited, and is exemplified by, for example, 0.001 mL/s or more per 1 cm ² of opening area. It may be 0.01 mL/s or more, 0.03 mL/s or more, 0.05 mL/s or more, 0.07 mL/s or more, and the upper limit is, for example, 20 mL/s.

Further, the liquid feeding can be performed in a sterile environment. In the present invention, if the recess can be filled with a liquid, the cell culture substrate may be subjected to known treatments required for cell culture (for example, exchanging the culture medium in a safety cabinet, standing still in an incubator) as necessary. It's okay.

In this way, it is possible to manufacture a container for cell culture filled with a culture solution in a state in which at least air bubbles are difficult to form or remain in the recesses. In addition, in the present invention, even if the bubbles are formed by filling a liquid in a place where no liquid exists, or the bubbles are formed after being left for a while after filling, it is possible to remove the bubbles.

The obtained cell culture container is highly convenient because air bubbles can be visually confirmed, and since air bubbles are less likely to exist on the culture surface, the cells and spheroids obtained by culturing are highly homogeneous. be. Therefore, the present invention also provides a method for culturing cells and a method for producing spheroids using the cell culture container produced by the above method. That is, the cell culturing method and the spheroid manufacturing method of the present invention are not particularly limited as long as they include at least the above-described liquid feeding step. In the cell culturing process and the spheroid culturing process, settings can be made as appropriate according to known techniques.

In addition, the liquid feeding in the present invention makes it difficult for air bubbles to form or remain in the recess and allows the culture solution to be filled. A liquid feeding solution is fed from a liquid feeding port having a diameter of 3 mm or less and/or at a flow rate of 100 cm/s or more to a cell culture substrate having a cell culture substrate and a cell culture container in which a culture medium is provided in at least the recess. A method for defoaming a cell culture container containing a culture solution can be provided. The liquid feeding method and the applicable substrate can be determined with reference to the method for manufacturing a cell culture container of the present invention.

EXAMPLES Hereinafter, the present invention will be explained in detail with reference to Examples, but the present invention is not limited thereto. In addition, in the following examples, room temperature means 20 to 30°C.

Manufacturing example 1
<Preparation of layer with cell adhesive surface (preparation of fluorine-containing polyimide film)>
In a 500 mL three-necked flask, 25.332 g (0.057 mol) of 4,4'-hexafluoroisopropylidene diphthalic anhydride and 166.60 g of N-methylpyrrolidone were charged and dissolved. A solution of 16.668 g (0.049 mol) of 1,4-bis(aminophenoxy)benzene dissolved in 71.4 g of N-methylpyrrolidone was added dropwise thereto, stirred at room temperature under nitrogen atmosphere, and kept for 5 days. In this way, a fluorine-containing polyamic acid resin composition (solid content concentration 15% by mass, 6FDA/TPEQ polyamic acid) was obtained. The weight average molecular weight of the polyamic acid was 153,000, and the viscosity was 6.7 Pa·s. Note that the weight average molecular weight of the polyamic acid and the weight average molecular weight of the fluorine-containing polyimide after firing are substantially the same.

The fluorine-containing polyamic acid resin composition obtained above was applied onto a glass substrate using a die coater so that the thickness of the fluorine-containing polyimide film after firing was 40 μm to form a coating film. Next, the coating film was baked at 360° C. for 1 hour in a nitrogen atmosphere. Thereafter, the fired product was peeled from the glass substrate to obtain a fluorine-containing polyimide film. This fluorine-containing polyimide film had a static water contact angle of 83.2° and a falling angle of 26.4°.

The method for measuring the physical properties in the above is as follows.
(Measurement of weight average molecular weight)
Equipment: HCL-8220GPC manufactured by Tosoh Corporation
Column: TSKgel Super AWM-H
Eluent (LiBr・H2O, NMP with phosphoric acid): 0.01 mol/L
Measurement method: A 0.5% by weight solution is prepared using an eluent, and the molecular weight is calculated based on a calibration curve prepared with polystyrene.
(Measurement of viscosity)
Equipment: AS ONE Viscometer VISCOMETER TV-22
Setting: VI RANGE: H ROTOR No.6 SPEED: 10rpm
Standard solution for viscometer calibration: Nippon Grease Co., Ltd. JS 14000
Measurement method: After calibrating with the viscometer calibration standard solution, measure using 0.3g of varnish. (Measurement temperature: 23℃)
(Measurement of static water contact angle)
Equipment: Automatic contact angle meter (Kyowa Interface Science: DM-500)
Measurement method: Immediately after dropping 2 μL of water onto a surface (cell non-adhesive surface or cell adhesive surface) or film (film formed from a cell non-adhesive or cell adhesive substance), measure the adhesion angle of the droplet. Measure (measurement temperature: 25°C).
(Measurement of falling angle)
Equipment: Automatic contact angle meter (Kyowa Interface Science: DM-500)
Measurement method: After dropping 25 μL of water onto the surface (cell non-adhesive surface or cell adhesive surface) or film (film formed from a cell non-adhesive or cell adhesive substance), the sheet is continuously tilted. The angle at which it flows down is defined as the falling angle (measurement temperature: 25°C).

<Preparation of layer with non-cell adhesive surface>
After peeling the release tape on one side of the double-sided tape (thickness 25 μm) and bonding it to a transparent PET film (thickness 250 μm), use a CO ₂ laser to create through holes in a staggered arrangement with a diameter of 300 μm and a pitch of 500 μm. (Through holes formed: 400 holes/cm ² , 42000 holes/sheet, hole diameter on the laser incidence side: 500 μm, hole diameter on the laser emission side: 300 μm). Then, using a spin coater (MS-A150 manufactured by Mikasa), coat the PET film side surface with MPC polymer solution (0.5% ethanol solution) to a thickness of 0.05 μm, and dry in a dryer at 50°C for 2 A drying process was performed for a period of time to obtain a layer having a cell-nonadhesive surface [static water contact angle of 107.5° on the coating layer (MPC polymer coating layer) side of the PET film].

<Preparation of cell culture sheets/cell culture substrates>
Next, the layer having a cell adhesive surface prepared above was attached to the side from which the other release tape of the double-sided tape of the layer having a cell non-adhesive surface was removed, to prepare a cell culture sheet. (Sheet thickness: 315μm). The obtained cell culture sheet was placed in a culture plate to complete a cell culture substrate for use in cell culture.

Example 1
Defoaming treatment was performed using the cell culture substrate prepared above. Specifically, in a safety cabinet, approximately 20 mL of PBS was first added by decant to the cell culture substrate (the condition of the culture container at this time was the non-defoamed state shown in Figure 1, and approximately 99% of the wells were bubbles were observed). Next, fill a 20mL all-plastic (manufactured by Nipro) with 20mL of PBS to which an 18G non-bevel needle (inner diameter 0.9mm, manufactured by Terumo) is attached, and place the tip of the needle in the PBS liquid in the container (above the opening of the recess). By extruding the syringe while moving it horizontally at a position approximately 2 mm above the end surface, the entire amount of liquid is delivered to approximately 1/3 of the entire surface of the base material in approximately 8 seconds (approximately 0.09 mL/s per 1 cm ² of opening area, After the flow rate was approximately 393 cm/s), the PBS in the container was sucked up with a syringe, the syringe was refilled with PBS, and the same liquid feeding was repeated to the remaining area to degas it. (Defoamed state in Figure 1). Although air bubbles were no longer observed in most of the wells, defoaming was performed in the same manner as above for areas where air bubbles remained. By this treatment, almost all air bubbles could be removed. Thereafter, the tube was allowed to stand in a 5% (v/v) CO ₂ incubator at 37° C. for 15 minutes, and then the PBS filled in the syringe was extruded again to remove newly generated air bubbles. After such defoaming, remove the PBS so that it does not completely disappear from the well, then add 21 mL of KBM ADSC-2 medium containing 1% antibiotics, and add 21 mL of KBM ADSC-2 medium containing 1% antibiotics to 5% (v/v) at 37°C. v) The culture vessel was left standing overnight in a CO ₂ incubator to obtain a culture vessel having a culture substrate in which the recesses were filled with a culture solution.

Test example 1
<Cell expansion culture>
The cells used were human adipose derived stem cells (AdSC). AdSC was purchased from a manufacturer (Lonza, PT-5006) and used.

Frozen cells were lysed in a thermostatic water bath at 37°C and added to 9 mL of KBM ADSC-2 medium (basal medium, manufactured by Kohjin Bio) containing 5% FBS and 1% antibiotics. After centrifugation at 210×g for 5 minutes, the supernatant was removed and dispersed in 1 mL of basal medium. Add the basal medium in advance so that the total volume after adding the cell suspension to an 800 mL cell culture flask with a filter cap (manufactured by Sumitomo Bakelite) is 30 mL, and add the cell suspension to 1.5 × 10 ⁶ cells. / flask, and cultured (expanded culture) in a 5% (v/v) CO ₂ incubator at 37°C.

<Preparation of spheroids>
The medium was removed from the culture flask, 5 mL of cell detachment solution accutase (manufactured by Promocell) was added, and the cells were detached by holding in a 5% (v/v) CO ₂ incubator at 37° C. for about 5 minutes. Next, the stripping solution was collected and transferred to a tube using PBS in a total volume of 15 mL. The cells were centrifuged at 210×g for 5 minutes, suspended in 4 mL of KBM ADSC-2 medium containing 1% antibiotics, and the number of cells was counted. Thereafter, the concentration was adjusted to 1.0×10 ⁶ cells/mL.

The medium was removed from the culture container prepared in Example 1, and cells were seeded at 500 cells/well. After standing in a safety cabinet for 15 minutes, the cells were placed in a 5% (v/v) CO ₂ incubator at 37°C and cultured for 3 days (Figure 2).

As a result, in the case of non-defoaming, residual air bubbles were observed here and there, but no air bubbles were observed in the recesses of the culture vessels obtained by the method of the present invention (FIG. 1). Furthermore, by using the culture container obtained by the method of the present invention, spheroids could be produced in almost all cavities (FIG. 2). When the container was photographed using a plate scanner (manufactured by Screen) and the resulting image was divided into 9 parts to calculate the spheroid production efficiency at each point, the spheroid production efficiency was 99 ± 1% (average value of 9 locations). there were.

Example 2
A culture vessel was prepared with reference to Example 1, except for using NanoCulture Plate MS Pattern High-Binding 24 well (manufactured by SCIVAX Life Sciences), which has multiple recesses with an opening diameter of 2.5 μm on the culture surface of each well. Obtained. Specifically, a 20 mL all-plastic (manufactured by Nipro) equipped with an 18G non-bevel needle (inner diameter 0.9 mm, manufactured by Terumo) is filled with PBS, and in each well, the tip of the needle is approximately 2 mm above the upper end surface of the recess opening. At this point, 20 mL of liquid was delivered in about 8 seconds (approximately 0.09 mL/s per 1 cm ² of opening area, flow rate of approximately 393 cm/s) to defoam.

Example 3
A culture vessel was prepared with reference to Example 1, except for using NanoCulture Plate MS Pattern Low-Binding 24 well (manufactured by SCIVAX Life Sciences), which has multiple recesses with an opening diameter of 2.5 μm on the culture surface of each well. Obtained. Specifically, a 20 mL all-plastic (manufactured by Nipro) equipped with an 18G non-bevel needle (inner diameter 0.9 mm, manufactured by Terumo) is filled with PBS, and in each well, the tip of the needle is approximately 2 mm above the upper end surface of the recess opening. At this point, 20 mL of liquid was delivered in about 8 seconds (approximately 0.09 mL/s per 1 cm ² of opening area, flow rate of approximately 393 cm/s) to defoam.

Test example 2
The culture vessels of Example 2 and Example 3 were subjected to microscopic observation in comparison with the case where the culture medium was filled with the culture medium before defoaming treatment (non-defoaming). The areas where bubbles remain appear black, and the areas where bubbles do not remain appear white.

It was confirmed that bubbles were not easily formed in the 24-well plates of Example 2 (Figure 3) and Example 3 (Figure 4), and defoaming was possible even in containers with concave portions smaller in diameter than in Example 1. In addition, defoaming was possible regardless of the material of the culture surface. Conventionally, defoaming was done by centrifugation, but by using this method, defoaming could be done without removing the container from the sterile environment.

By the method of the present invention, it is possible to easily prepare a cell culture container that is not easily affected by air bubbles, and the cell culture work itself can be performed efficiently. It can be suitably used in the field of pharmaceutical preparations.

Claims (14)

A method for manufacturing a cell culture substrate having a concave portion with an opening diameter of 1000 μm or less and a cell culture container in which a culture solution is provided at least in the concave portion, the method comprising After injecting into the culture substrate, from 0.1 to 10 mm above the recess, from the liquid feeding port with a diameter of 3 mm or less , at a flow rate of 100 cm/s or more and 0.001 mL/s or more per 1 ^cm2 of opening area. A method for manufacturing a cell culture container, comprising the step of feeding a liquid feeding liquid at a flow rate to a position immersed in the injected liquid . A defoaming method for a cell culture substrate having a recess with an opening diameter of 1000 μm or less and a cell culture container in which a culture solution is provided at least in the recess, the method comprising: pre-filling a cell culture container with a culture solution before sending the liquid; After injecting into the cell culture substrate, from 0.1 to 10 mm above the recess, from the liquid feeding port with a diameter of 3 mm or less , at a flow rate of 100 cm/s or more and 0.001 mL/s or more per 1 cm ² of opening area. A defoaming method for a cell culture container in which a liquid is delivered to a position immersed in the injected liquid at a flow rate of . 3. The method according to claim 1, wherein the liquid is fed from a liquid feeding port having a diameter of 2.5 mm or less. The method according to any one of claims 1 to 3, wherein the liquid is fed at a flow rate of 200 cm/s or more. The method according to any one of claims 2 to 4 , comprising the step of leaving the cell culture container stationary in a CO ₂ incubator. The method according to any one of claims 1 to 5, wherein the liquid is delivered at a flow rate of 0.01 mL/s or more per 1 cm ² of opening area. A flow rate of 200 cm/s or more from a liquid feeding port with a diameter of 2.5 mm or less, and an opening area of 1 cm. ² 7. The method according to claim 1, wherein the liquid is fed at a flow rate of 0.01 mL/s or more. From a liquid feeding port with a diameter of 2.0 mm or less, a flow rate of 250 cm/s or more and an opening area of 1 cm. ² 8. The method according to claim 1, wherein the liquid is fed at a flow rate of 0.03 mL/s or more. 9. The method according to claim 1, wherein the ratio of the diameter of the liquid feeding port to the diameter of the opening of the recess (diameter of liquid feeding port/diameter of the opening of the recess) is 30 or less. The cell culture substrate has a plurality of recesses each having an opening diameter of 1000 μm or less, the inner surface of the recess has a cell non-adhesive surface, and the bottom surface of the recess has a cell adhesive surface. The method according to any one of claims 1 to 9, which is a cell culture sheet. The method according to any one of claims 1 to 10, which is carried out in a sterile environment. The method according to any one of claims 1 to 11, wherein the liquid feeding liquid is injected into the cell culture substrate in advance before the liquid feeding. A method for culturing cells, comprising manufacturing or defoaming a cell culture container by the method according to any one of claims 1 to 12, and using the obtained cell culture container. A method for producing spheroids , comprising producing or defoaming a cell culture container by the method according to any one of claims 1 to 12, and using the obtained cell culture container.