JP7263512B2 - Method for collecting cells and/or aggregates thereof - Google Patents

Description

The present invention relates to a method for collecting cells and/or aggregates thereof.

Conventionally, various studies have been made on the recovery of cultured cells.

For example, Patent Document 1 discloses a method of using a medium containing cellulose nanofibers during cell culture to collapse the network of cellulose nanofibers formed in the medium by applying a light impact or the like, thereby sedimenting the cultured cells. disclosed.

Further, Patent Document 2 discloses a method for recovering cells in a sheet form by seeding cells on a temperature-responsive polymer to form a cell sheet, and then exfoliating the cells by giving a low temperature treatment stimulus. ing.

WO2017/199737 JP 2015-119694 A

However, in the methods of the prior art, in addition to requiring complicated preparations such as the need for a specific container and, in some cases, dedicated equipment for recovery, it also takes time to recover in the case of low-temperature treatment. Not enough yet. In addition, if a strong force is applied during recovery, the cells may be damaged, and the recovery efficiency may be low. Therefore, a further improved method is desired.

An object of the present invention is to provide a novel method for collecting cells and/or aggregates thereof, and a cell population collected by the method.

Another object of the present invention is to provide a simple and efficient method for collecting cells and/or aggregates thereof.

As a result of intensive studies to achieve the above object, the present inventors have found that, in a culture container having a culture compartment (well) with a small diameter, a liquid can be stably fed from a member having a specific diameter to the compartment. The inventors have found that cells can be collected simply and efficiently, and have completed the present invention through further studies.

That is, the present invention relates to the following [1] to [10].
[1] A liquid is fed to a cell culture substrate containing cells in a recess with an opening diameter of 1000 μm or less from a liquid feed port with a diameter of 3 mm or less and/or at a flow rate of 100 cm / s or more. A method for collecting cells and/or aggregates thereof, comprising a step of feeding a solution.
[2] The method according to [1] above, wherein the liquid is fed at a flow rate of 0.001 mL/s or more per 1 cm ² of opening area.
[3] The method according to [1] or [2] above, wherein the liquid is fed from 0.1 to 10 mm above the recess.
[4] The method according to any one of [1] to [3] above, wherein the diameter of the liquid feed port is 2.5 mm or less.
[5] Any one of [1] to [4] above, wherein the ratio of the diameter of the liquid feed port to the diameter of the opening of the recess (diameter of the liquid feed port/diameter of the opening of the recess) is 30 or less. the method of.
[6] The cell culture substratum has a plurality of recesses with a pore size of 1000 μm or less in diameter, the inner surfaces of the recesses have cell non-adhesive surfaces, and the bottom surfaces of the recesses are cell adhesive. The method according to any one of [1] to [5] above, which is a cell culture sheet having a surface.
[7] The method according to any one of [1] to [6] above, which is carried out in an aseptic environment.
[8] The method according to any one of [1] to [7], further comprising a step of recovering the liquid present on the substrate after the liquid transfer step.
[9] The method according to any one of [1] to [8] above, wherein the cells form spheroids.
[10] A cell population having a viable cell rate of 80% or more, obtained by the method according to any one of [1] to [9] above.

INDUSTRIAL APPLICABILITY According to the present invention, cells and/or aggregates thereof can be collected simply and efficiently.

FIG. 1 is a photograph of a culture vessel before collecting spheroids in Example 1. FIG. FIG. 2 is a photograph of the culture vessel after collecting spheroids in Example 1. FIG.

The method for recovering cells and/or aggregates thereof of the present invention is performed through a liquid feed opening having a diameter of 3 mm or less, and/or It includes a step of feeding the liquid for liquid feeding at a flow rate of 100 cm/s or more. Through such a step, the cells and/or aggregates existing in the recesses of the cell culture substrate may be exfoliated from the recesses or released to the outside of the recesses, and can be collected as they are.

The recovery method of the present invention can be applied to any substrate for cell culture that has on its surface concave portions with openings having a diameter of 1000 μm or less, as long as cells can be cultured in the concave portions.

The diameter of the concave portion is not particularly limited as long as the diameter is 1000 μm or less, 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. Also, the lower limit is not particularly set, and for example, 10 μm can be mentioned. Note that the diameter of the recess means 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 , 1.0×10 −1 cm 2 , 5.0} ×10 ⁻² cm per recess. ² , 2.0× ^10-2 cm2 ^, 1.0× ^{10-2 cm2 ,} 5.0× ^10-3 cm2, 2.5× ^10-3 cm2 ^, 2.0× ^{10-3 cm2} , 1.0× ^10-3 cm2 ^, etc. are exemplified.

The depth of the recess is not particularly limited as long as the cells can be cultured, and can be appropriately set according to the common general technical knowledge 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 (e.g., 100 nm or more), etc., and within the range of 1 μm or more [e.g., 5 μm or more, 10 μm or more (e.g., 10 to 1000 μm, 10 to 300 μm)] There may be.

The shape of the opening and the bottom (bottom) of the recess is not limited to circular, and may be, for example, polygonal or elliptical, and the shape of the bottom may be the same as or different from the shape of the opening. good. Also, the bottom surface may be flat (flat), curved, or smooth, but a flat and/or smooth bottom surface is more desirable. Examples of the area of the bottom surface per recess are within the range of 0.5×10 ⁻³ to 5.0×10 ⁻¹ cm ² , such as 1.0×10 ⁻¹ cm ² , 5.0×10 ⁻² cm ² , and 2.0×10 −1 cm 2 . ×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 and the like are exemplified. be. In addition, the diameter (length) of the bottom may be the same as or different from the diameter of the opening, and even if the diameter of the bottom is smaller than the diameter of the opening, may be larger than the diameter of the Examples of the diameter of the bottom surface are within the ranges 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 columnar. 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 hole diameter to the opening hole diameter (bottom hole diameter/opening hole diameter) is not particularly limited, but from the viewpoint of ease of cell seeding and recovery, it is 5/1 to 1/ 5, 3/1 to 1/3, 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, or 500 μm depending on the desired mass culture. Hereinafter, ranges such as 300 μm or less, 200 μm or less, 100 μm or less are exemplified, and any finite value is acceptable.

The ratio of the pore diameter of the opening to the depth of the recess (pore diameter of the opening/depth of the recess) is not particularly limited, but from the viewpoint of ease of cell seeding and recovery, it is from 5/1 to 1/5, 3/1 to 1/3, 2/1 to 1/1 are exemplified. Within the above range, it is difficult for cells to jump out of the recesses, and it can be advantageous in terms of cell recovery.

The number of concave portions cannot be generally set according to the area of the base material, the type of cells to be cultured and/or aggregates thereof, and the like, and can be appropriately set according to the common general technical knowledge. For example, the lower limit number per unit area (cm ² ) is 1, 10, 30, 50, etc., and the upper limit number is 1000, 500, 300, 200, 100, etc. . In addition, the total number of recesses on the base material surface can be appropriately set, for example, 10 or more, 100 or more, 1000 or more, 10000 or more, 50000 or more.

The material of the cell culture substrate is not particularly limited as long as it is known. Further, 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 concave portion and the surface of the substrate may be made of different materials, and furthermore, different materials may be used depending on the position in the concave portion.

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. A cell culture sheet having Such a cell culture sheet has the above-described surface characteristics in addition to the small diameter of the openings, which makes it easier for the culture to adhere to the substrate, making recovery more difficult. It is possible to obtain high recovery efficiency while reducing damage.
The method for collecting cells and/or aggregates thereof of the present invention can be applied to various cell culture substrates, but is preferably used for collecting cells cultured on the cell-adhesive surface of the substrate. More preferably, it is used for recovering cells adherently cultured on a surface exhibiting moderate cell adhesiveness.

The term "cell-adhesive surface" refers to a surface on which, when cells are sedimented on the surface in a solution used for culture, the cells adhere to each other with certain adhesion points. . The cell-adhesive surface may be composed of at least a substance exhibiting cell-adhesiveness.

The substance exhibiting cell adhesion is not particularly limited 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 hydrophilic or hydrophobic, but from the viewpoint of cell adhesiveness, spheroid formation, etc., hydrophilicity (especially hydrophilicity that is not superhydrophilicity) or hydrophobicity (Particularly, hydrophobicity that is not superhydrophobicity) is preferred, and one that exhibits hydrophobicity is more preferred. Also, the degree of adhesiveness of the substance exhibiting cell adhesiveness may be such that the cells do not protrude from the concave portion.
Specific examples of such substances include substances obtained from living organisms or synthesized, for example, proteins (collagen, fibronectin, laminin, etc.), synthetic resins (styrene resin, polyolefin resin , polycycloolefin resins, polyethylene terephthalate resins, acrylic resins, fluorine resins, polyimide resins, polysulfone resins, polyethersulfone resins, polydimethylsiloxane resins, mixtures thereof, surface-modified materials, etc.). When a synthetic resin is selected, a cell culture sheet with excellent handleability 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 a synthetic resin such as a styrene resin, a polyolefin resin, or a polyimide resin. By selecting non-biological components such as styrene resins, polyolefin resins, and polyimide resins, spheroids obtained using cell culture substrates containing styrene resins, polyolefin resins, polyimide resins, etc. are useful for regenerative medicine. It is easy to apply to fields such as drug discovery and drug discovery.

Examples of polyimide resins include polyimide resins containing structural units represented by the following formula (I). Moreover, 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 imidating polyamic acid obtained by polymerizing one or more of each of acid dianhydride and diamine. The polyimide resin may contain polyamic acid as part of its chemical structure. As a method for producing a polyimide resin, it may be produced by a known method. As an example, a two-step synthesis method can be used. The two-step synthesis method of polyimide resin is a method of synthesizing polyamic acid as a precursor and converting polyamic acid into polyimide. Polyamic acid as a precursor may be a polyamic acid derivative. Examples of polyamic acid derivatives include polyamic acid salts, polyamic acid alkyl esters, polyamic acid amides, polyamic acid derivatives from bismethylidenepyromeritide, polyamic acid silyl esters, and polyamic acid isoimides. As polyimides, acid anhydrides such as pyromellitic dianhydride, biphenyltetracarboxylic dianhydride, and benzophenonetetracarboxylic dianhydride, and diamines such as oxydiamine, paraphenylenediamine, metaphenylenediamine, and benzophenonediamine. Polyimide can be exemplified. Examples of resins having fluorine atoms 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'-isopropylidenedi phenoxy)diphthalic acid (BPADA)/2,2-bis[4-(4-aminophenoxy)phenyl]hexafluoropropane (HFBAPP), 6FDA/2,2-bis(4-(4-aminophenoxy)phenyl)propane Fluorine-containing polyimide resins containing structural units represented by the following formula (I) such as (BAPP) copolymers; ethylene-tetrafluoroethylene copolymers and the like can be exemplified.

In formula (I) above, X ⁰ represents either 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 structural unit of the resin, or may be the same. At least one of X ⁰ , Y, Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ and Z ⁶ preferably 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 connected through ^X0 .

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. It may also be a condensed cyclic bivalent hydrocarbon group, a heterocyclic condensed cyclic bivalent hydrocarbon group, and an oxy group and a thio group thereof. Among these, an alkylene group, an aryleneoxy group, and an arylenethio group are preferable, and an alkylene group and an aryleneoxy group are more preferable, and these may be substituted with a fluorine atom. The number of carbon atoms in the alkylene group is, for example, 1-12, preferably 1-6.

Examples of fluorine atom-substituted alkylene groups for X ⁰ include -C(CF ₃ ) ₂ -, -C(CF ₃ ) ₂ -C(CF ₃ ) ₂ -, and the like. . Among the above-described alkylene groups that are examples of X ⁰ , -C(CF ₃ ) ₂ - is preferred.

Examples of arylene groups that are examples of X ⁰ include the following.

Examples of aryleneoxy groups that are examples of X ⁰ include the following.

Examples of arylene thio groups that are examples of X ⁰ include the following.

From the viewpoint that spheroids can be well formed 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. may be selected from the group consisting of b-7 to b-9 and c-7 to c-9 above, or may be a structure represented by b-8.

The above-mentioned arylene group, aryleneoxy group and arylenethio group which are examples of X ⁰ are each independently a halogen atom (e.g., fluorine atom, chlorine atom, bromine atom, iodine atom, preferably fluorine atom or chlorine atom, more preferably fluorine atom), may be substituted by a group selected from the group consisting of a methyl group and a trifluoromethyl group. A plurality of these substituents may be used, and in that case, the types of substituents may be the same or different. Preferred substituents on arylene groups, aryleneoxy groups and arylenethio groups are fluorine atoms and/or trifluoromethyl groups, preferably fluorine atoms. The arylene group, aryleneoxy group and arylenethio group are preferably substituted with at least one or more fluorine atoms 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 (that is, a single bond or an alkylene group), an oxygen atom, a sulfur atom, or directly mentioned. Specifically, the following groups can be exemplified.

The divalent organic group having an aromatic ring described above, which is an example of Y, is, if substitutable, a halogen atom (e.g., fluorine atom, chlorine atom, bromine atom, iodine atom, preferably fluorine atom or chlorine atom , more preferably a fluorine atom), may be substituted with a group selected from the group consisting of a methyl group and a trifluoromethyl group. A plurality of these substituents may be used, and in that case, the types of substituents may be the same or different. Preferred substituents on the divalent organic group having an aromatic ring are preferably fluorine atoms and/or trifluoromethyl groups, particularly when X ⁰ does not contain a fluorine atom, and more preferably is a fluorine atom.

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

In formula (I) above, 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, a fluorine atom, , a chlorine atom, a bromine atom or an iodine atom, and at least one of Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ and Z ⁶ when at least one of X ⁰ and Y does not contain a fluorine atom is preferably a fluorine atom.

From the viewpoint of spheroid formation, in a preferred embodiment of the present invention, the divalent organic group represented by X ⁰ in formula (I) is —C(CF ₃ ) ₂ —, b-2 to b -10 and c-2 to c-10; and Y is 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.

A polyimide resin composed of structural units represented by the above formula (I) can be obtained by a method of baking a polyamic acid obtained by polymerization of an acid dianhydride and a diamine. It should be noted that the imidization rate of the "polyimide resin comprising the structural unit represented by formula (I)" does not have to be 100%. That is, the polyimide resin composed of the structural unit represented by formula (I) may be composed only of the structural unit represented by formula (I), as long as the object and effect of the present invention are not impaired. , may partially contain a structural unit in which the cyclic imide structure remains an amic acid without dehydration and ring closure.

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 the reaction between the acid dianhydride and the diamine, which are raw materials, can proceed efficiently and is inert to these raw materials. . For example N-methylpyrrolidone (NMP), N,N-dimethylacetamide, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, sulfolane, methyl isobutyl ketone, acetonitrile, benzonitrile, nitrobenzene, nitromethane, acetone, methyl ethyl ketone, isobutyl ketone , polar solvents such as methanol; and non-polar solvents such as toluene and xylene. Among them, 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. Although the concentration of the polyamic acid in the polyamic acid solution is not particularly limited, 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. Although the viscosity of the resin composition is not particularly limited, it can be measured according to a known measuring method. .

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 (thermally imidized) by heat treatment to obtain a resin containing a fluorine-containing polyimide. Polyimides obtained by thermal imidization eliminate the possibility of residual catalyst and are more preferred for cell culture applications.

In the case of imidization by thermal imidization, for example, the polyamic acid is heated in air, or more preferably in an inert gas atmosphere such as nitrogen, helium, argon, etc., or in vacuum, preferably at a temperature of 50 to 400 ° C. , more preferably 100 to 380° C., preferably for 0.1 to 10 hours, more preferably 0.2 to 5 hours, to carry out the imidation reaction to obtain a resin composition containing polyimide.

The polyamic acid to be subjected to the thermal imidization reaction is preferably dissolved in a suitable solvent. Any solvent can be used as long as it dissolves the polyamic acid, and the above solvents for 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 an appropriate solvent.

The dehydration cyclization reagent can be used without particular limitation as long as it has the effect of chemically dehydrating cyclodehydration of polyamic acid to form a polyimide. As such a dehydration cyclization reagent, use of a tertiary amine compound alone or a combination of a tertiary amine compound and a carboxylic acid anhydride can efficiently promote imidization. is preferred.

Examples of tertiary amine compounds 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. Only one kind of tertiary amine may be used, or two or more kinds thereof may be used.

Examples of carboxylic anhydrides include acetic anhydride, trifluoroacetic anhydride, propionic anhydride, butyric anhydride, isobutyric anhydride, succinic anhydride, and maleic anhydride. Among these, acetic anhydride and trifluoroacetic anhydride are particularly preferred, and acetic anhydride is more preferred. Only one kind of carboxylic anhydride may be used, or two or more kinds thereof may be used.

As a solvent for dissolving polyamic acid in chemical imidization, a polar solvent having 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 It is preferably one or more selected from the group consisting of -methylpyrrolidone from the viewpoint of homogeneous reaction. When these solvents are used as the solvent for the amidation reaction, the reaction mixture after the amidation reaction can be directly used for chemical imidization without separation of the polyamic acid.

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, more preferably 20,000 to 500,000. In the present specification, the weight-average molecular weight of the resin can be measured according to a known measurement method, and the weight-average molecular weight in the above range makes it easier to synthesize and handle polyimide resins, form films, and form spheroids. become good.

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

Substances or surfaces exhibiting cell adhesiveness [hydrophobic (particularly non-superhydrophobic) hydrophobic substances or surfaces exhibiting cell adhesion] must have a static water contact angle of 70° or more. The slip angle may be 15° or more, or the static water contact angle may be 70° or more and the slip angle may be 15° or more.
In particular, a substance or cell-adhesive surface that exhibits cell-adhesiveness [a hydrophobic (particularly hydrophobic, non-superhydrophobic) cell-adhesive substance or cell-adhesive surface] has a static water contact angle of 70° or more. is preferably Spheroid formation is further promoted when the cell-adhesive surface satisfies such conditions. From the viewpoint of spheroid formation, the static water contact angle is more preferably greater than 75°, still more preferably 77° or greater, even more preferably 79° or greater, and particularly preferably 80° or greater (e.g., greater 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, particularly 100° or less (e.g., 99 °, 98° or less, 97° or less, 95° or less, etc.), and more preferably less than 90°.

On the other hand, the static water contact angle of the cell-adhesive substance or cell-adhesive surface [hydrophilic (particularly non-super-hydrophilic) cell-adhesive substance or cell-adhesive surface] is preferably 65. ° or less, more preferably 55° or less, and even more preferably 50° or less. The lower limit may be 0° or higher, preferably 5° or higher, and more preferably 10° or higher.
From the same point of view, the falling angle of the substance exhibiting cell adhesiveness or the 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 higher, the higher the better. The upper limit of the falling angle is, for example, less than 80°, preferably 70° or less (eg, less than 70°), more preferably 60° or less (eg, less than 60°), and still more preferably 50° or less ( less than 50°).
Such a static water contact angle or sliding angle may be a value for a cell-adhesive surface, or a value for a substance exhibiting cell-adhesiveness (or a substance constituting a cell-adhesive surface). good.
The surface properties can be measured according to known methods. For example, the static water contact angle and the falling angle may be measured by, for example, the method described later.

When the surface exhibiting cell adhesiveness constitutes the bottom surface of the recess, the ratio of the substance exhibiting cell adhesion to the bottom surface of the recess is not particularly limited, but 90% or more and 95% of the bottom surface of the recess. As mentioned above, it is preferable to occupy 99% or more, substantially all of the bottom surface. In addition, the cell-adhesive surface on the bottom surface is preferably flat (flat-bottomed, flat-bottomed) from the viewpoint of observability.

The term "cell non-adhesive surface" means that, for example, in a solution used for culture, when cells settle on the surface, the cells hardly change their shape, do not adhere at all, or temporarily It is a surface that spontaneously detaches even if it adheres weakly. The cell non-adhesive surface should be composed of at least a substance exhibiting cell non-adhesiveness.

The substance exhibiting cell non-adhesiveness is not particularly limited as long as it does not adhere to the cells used for culture or does 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.
In addition, the substance exhibiting cell non-adhesiveness (or cell non-adhesive surface) may exhibit hydrophobicity or hydrophilicity. or superhydrophilic. From the viewpoint of cell non-adhesiveness, spheroid uniformity and formation, etc., a hydrophobic (especially superhydrophobic) [e.g., a hydrophobic or hydrophilic (especially hydrophobic) cell-adhesive surface or the surface Substances that exhibit more hydrophobicity (especially superhydrophobicity) to the resin (and also its contact angle)] are particularly preferred, but hydrophilicity (especially superhydrophilicity) [e.g. hydrophilic or hydrophobic (e.g. ) or a substance that exhibits more hydrophilicity (especially superhydrophilicity) with respect to the cell-adhesive surface of ) or the resin that constitutes the surface (and the contact angle thereof)].
Specific examples of such substances include (poly)ethylene glycol and its derivatives, MPC (2-methacryloyloxyethylphosphorylcholine) and its derivatives, HEMA (hydroxyethyl methacrylate) and its derivatives, and the like. Compounds containing or polymers of those compounds [e.g., poly-HEMA (polyhydroxyethyl methacrylate), etc.], compounds such as SPC (segmented polyurethane) and its derivatives, proteins obtained from living organisms (albumin, etc.), cells Non-adhesive sugar chains (agarose, cellulose, etc.) can be appropriately selected and used according to the cell type. Among them, from the viewpoint of adhesion to cell adhesive surfaces and synthetic resins, from the viewpoint of simplifying the manufacturing process of cell culture substrates or cell culture sheets, or from the viewpoint of uniformity of cultured cells or spheroids obtained From the viewpoint of improving the properties, MPC, derivatives thereof, or polymers thereof are preferred.
The substance exhibiting cell non-adhesiveness should be appropriately denatured according to the degree of handleability, desired hydrophobicity (e.g., superhydrophobicity) or hydrophilicity (e.g., superhydrophilicity). may For example, by subjecting a hydrophilic substance to a cross-linking treatment or the like, both hydrophilicity and low solubility in water may be achieved. In addition, the raw material (e.g., hydrophobic or hydrophilic) is appropriately subjected to hydrophobic treatment or hydrophilic treatment (e.g., introduction of a hydrophobic group or hydrophilic group) to obtain the desired hydrophobicity or hydrophilicity. Material may be obtained.

Immobilization of substances exhibiting cell non-adhesion can be achieved by drying a solution containing these substances on the substrate surface, by melting the substance and applying pressure, or by exposing the substance applied to the substrate to energy rays such as UV rays. A method of curing with , causing a chemical reaction (e.g., 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 substrate to form covalent bonds. Alternatively, the substance can be immobilized on the surface of the substrate by binding the thiol group of the substance to a metal (platinum, gold, etc.) thin film preliminarily formed on the substrate. The thickness for immobilization is not particularly limited, and is exemplified from 0.01 to 1000 μm.

When the surface exhibiting cell non-adhesiveness constitutes the inner surface of the recess, the ratio of the surface exhibiting cell non-adhesiveness to the inner surface of the recess is not particularly limited, but is such that the adherence of cultured cells is reduced. is preferably It preferably occupies 90% or more, more preferably 95% or more, and particularly preferably all of the area of the inner surface.
A surface exhibiting cell non-adhesiveness can be judged by using, for example, a static water contact angle as an indicator of its surface properties from the viewpoint of uniforming the size of cultured cells and improving circularity. . For example, in the case of a hydrophobic surface formed of the substance exhibiting cell non-adhesiveness, the static water contact angle is preferably 90° or more, more preferably 93° or more, and still more preferably 95° or more. Also, it may be 150° or less, preferably 130° or less, more preferably 120° or less.
On the other hand, when the surface is hydrophilic, the static water contact angle is preferably 65° or less, more preferably 55° or less, and even more preferably 50° or less. Moreover, it may be 0° or more, preferably 5° or more, and more preferably 10° or more.
To give an example of such a substance, a highly hydrophobic MPC (or a surface formed of the MPC) has a static water contact angle of, for example, 90° or more, 100° or more. can be realized.
Such a static water contact angle may be a value for a cell non-adhesive surface, or a value for a substance exhibiting cell non-adhesiveness (or a substance constituting a cell non-adhesive surface). good.
Also, the static water contact angle may be measured, for example, by the method described later.

From the viewpoint of improving the size uniformity and circularity of the resulting 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 cell-non-adhesive surface exhibits cell-adhesiveness, it may be less adhesive 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 the cell non-adhesive surface (or substance exhibiting cell non-adhesiveness) is 3° or more (e.g. , 5° or more), more preferably 10° or more (for example, 12° or more), and even more preferably 15° or more. In addition, the upper limit of the above difference (or its absolute value) is the hydrophobicity/hydrophilicity of the cell non-adhesive surface (or substance exhibiting cell non-adhesiveness) and the cell adhesive surface (or substance exhibiting cell adhesiveness). It can be selected as appropriate according to the combination, etc., and is not particularly limited.

The surfaces having these characteristics are independently attached to the base material surface such that the inner surface of the recess is a cell non-adhesive surface and the bottom surface of the recess is a cell adhesive surface. It may be formed by chemically fixing or arranging, or may be formed by a sheet-like member made of a substance having the relevant properties.

Further, in the cell culture sheet described above, the sheet surface, which is also the peripheral 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 such recesses may be a layered structure comprising a layer containing the bottom surfaces of the recesses and a layer containing the inner surfaces of the recesses. Here, the layer including the inner side surface of the concave portion constitutes a layer having a through hole. Therefore, one aspect 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 formed by fixing a substance exhibiting cell-non-adhesiveness to the layered base material from the viewpoint of forming the through-holes or from the viewpoint of simplifying the production of the cell culture sheet. A modified one is preferable.

As the layered base material, any one known in the art can be used. Synthesis of polystyrene, polyethylene, polypropylene, polycarbonate, polyamide, polyacetal, polyester (polyethylene terephthalate, etc.), polyurethane, polysulfone, polyacrylate, polymethacrylate, polycycloolefin, polyetherketone, polyetheretherketone, polyimide, silicone, etc. A plate-shaped body made of resin, synthetic rubber such as EPDM (ethylene propylene diene rubber), natural rubber, glass, ceramic, metal material such as stainless steel, or the like can be used. A transparent base material is also one of the preferred forms.

From the viewpoint of workability, the immobilization of the substance exhibiting cell non-adhesiveness to the layered base material is preferably carried out after forming through-holes, which will be described later.

The through-hole preferably has a wall corresponding to the inner side surface of the above-described recess, and the diameter and shape of the opening and the opposite end thereof can be set in the same manner as the above-described recess. Further, the depth of the through-holes corresponds to the thickness of the layer having the cell-non-adhesive surface, but it also corresponds to the depth of the recesses described above, and can be set in the same manner as the recesses. 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 layer of the substance exhibiting cell non-adhesiveness and the base material can be appropriately set as long as it falls within the thickness range of the layer having the cell non-adhesive surface.

Formation of the through-hole is not particularly limited as long as the through-hole having the size described above can be formed. For example, it can be formed by perforating (drilling, etc.), optical microfabrication (laser (eg, CO ² laser, excimer laser, semiconductor laser, YAG laser), etc.), etching, embossing, and the like. In the processing, the shape of the through-hole may be tapered. A structure may be formed in which the layer thickness is different in the portion located in the intermediate region.

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 recesses.

The cell culture sheet also includes an aspect in which an adhesive layer (adhesive layer) is further included between the layer having a cell-adhesive surface and the layer having a non-cell-adhesive surface.

Any adhesive layer known in the art can be used as the adhesive layer. Examples thereof include acrylic resins, silicone resins, synthetic rubbers, natural rubbers, etc. Preferably, low-eluting adhesive layers can be used. A commercially available double-sided tape or the like may 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, 0.5-100 μm is exemplified.

The cell culture sheet can be produced by laminating a layer having a cell non-adhesive surface having through holes and a layer having a cell adhesive surface in this order. 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 separate layer may be formed on a layer prepared in advance, or a combination of these may be used. good too. Specifically, for example, a substance exhibiting cell adhesiveness is cast, spray-coated, or coated on a release sheet (e.g., an organic polymer film such as a polyethylene base material, ceramics, metal, glass, etc.) whose surface has been release-treated. A layer having a cell-adhesive surface can be formed into a sheet by coating to an appropriate thickness by a method such as dip coating, spin coating, or roll coating and heating. On the other hand, a layer having a cell non-adhesive surface may be prepared in advance by coating the surface with a substance exhibiting cell non-adhesiveness after forming through holes in the base material of the layer having a cell non-adhesive surface. can be done. Then, it can be produced by laminating a separately prepared layer having a cell non-adhesive surface after peeling off the release sheet of the layer having a cell adhesive surface formed as described above. In the lamination of the layer having a cell non-adhesive surface and the layer having a cell adhesive surface, the above-described adhesive layer (adhesive layer) may be used for lamination, or welding (high frequency welding, ultrasonic welding) etc.), or may be laminated by pressure bonding (thermocompression bonding, etc.).

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

The substrate for cell culture such as the sheet for cell culture obtained in this way may be appropriately sized according to the size of the target device from the viewpoint of using it as it is installed in a known cell culture device. Cell culture may be performed by placing a medium containing cells on one surface thereof, and the cell culture substrate is used as a culture plate, each well of the plate, a culture petri dish (culture dish), a flask, a culture bag, or the like. Cell culture can be carried out on a part or the entire surface of the fixed cell culture substratum by accommodating and fixing it in various cell culture vessels.

The cells and/or aggregates collected from the recesses of the cell culture substratum may be cultured in suspension in the recesses or adherently cultured on the bottom surface of the recesses. The type is not particularly limited, for example, any organ or tissue (brain, liver, pancreas, spleen, heart, lung, intestine) of humans or non-human animals (monkeys, pigs, dogs, rabbits, rats, mice, etc.) , cartilage, bone, fat, kidney, nerve, skin, bone marrow, dental pulp, embryo, etc.), established cell lines, or genetically engineered cells can be used. The cell aggregate may be an aggregate (cell aggregate, spheroid) formed by culturing cells. The number of cells and/or aggregates thereof in a depression is not particularly limited, and is exemplified by 1 to 10,000 cells per depression. In addition, the size (diameter) of the collected cells and/or aggregates thereof is, for example, preferably 5-1000 μm, more preferably 10-500 μm.

As long as the cells and/or aggregates thereof can be cultured in the recesses, the amount of the culture solution in the entire substrate is not limited as long as at least the recesses are filled with the culture solution. In addition, when culturing cells and/or aggregates thereof in the recesses, the cell culture substrate may be subjected to defoaming treatment in advance, if necessary. The defoaming treatment is not particularly limited, and general treatment such as spraying, shaking, temperature change such as heating and cooling, centrifugal treatment, vacuum degassing, and ultrasonic treatment can be performed. Further, since the diameter of the opening of the recess of the cell culture substrate used in the present invention is 1000 μm or less, from the liquid feed port with a diameter of 3 mm or less and/or at a flow rate of 100 cm / s or more, The defoaming treatment may be performed by feeding the liquid for liquid feeding. By doing so, a series of steps from defoaming treatment to recovery can be performed efficiently. Therefore, as one aspect of the present invention, a cell culture substrate having a recess with an opening diameter of 1000 μm or less,
Cell culture in which a liquid for liquid feeding 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 degas, and the culture medium is provided to the substrate and at least the recess. preparing a container for
The step of culturing cells and/or aggregates thereof in the obtained cell culture vessel, and the cell culture vessel after the culture step, from a liquid feed port having a diameter of 3 mm or less and/or flow rate A method for collecting cells and/or aggregates thereof, which includes a step of collecting by feeding a liquid-feeding liquid at 100 cm/s or more, can be mentioned.

In the defoaming step, it seems that the more hydrophobic the material of the cell culture substrate is, the stronger the adherence of air bubbles becomes. can be done.

A device or instrument used for liquid transfer in defoaming has, for example, at least a liquid transfer port with a diameter of 3 mm or less. Examples of the liquid feeding port include those having a liquid feeding port with a diameter of 3 mm or less, and may have a diameter of 2.5 mm or less, 2.0 mm or less, or 1.7 mm or less, for example. In addition, from the viewpoint of the 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 opening of the recess) is It is preferably 30 or less, more preferably 20 or less, still more preferably 10 or less. The lower limit is not particularly limited, and may be, for example, 0.1 or more. In addition, the diameter of the liquid feeding port means the maximum inner diameter of the liquid feeding port.

The shape and material of the liquid feed port are not particularly limited as long as it has a diameter of the size described above. For example, it is possible to use those manufactured or adjusted to have the above-mentioned bore in the liquid delivery port of a known dispenser, injection needles, blunt needle cannulas, tips, sondes, etc. having the above-mentioned bore.

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

There are no particular restrictions on the liquid to be sent for defoaming. The culture medium itself may be used as the defoaming liquid-feeding liquid, or a physiological saline solution, a buffer solution, or the like may be used. When the culture medium is not used, the liquid filled in the concave portion is removed after the liquid is fed. Alternatively, the recesses of the cell culture substrate can be filled with the culture solution by adding the culture solution as it is after feeding the liquid. In addition, before the liquid feeding, the liquid for liquid feeding can be injected into the cell culture substrate in advance and then the defoaming treatment can be performed. may The pre-injection method and the amount of the liquid for liquid transfer are not particularly limited.

The method of sending the liquid for defoaming is not particularly limited as long as the liquid for sending the liquid can flow out from the liquid sending port and fill the concave portion. Specifically, for example, there is a method in which the tip of the liquid feed port is placed 0.1 to 10 mm above the upper end surface of the opening of the recess to feed the liquid. At that time, the tip of the liquid feeding port may be arranged so as to be immersed in the liquid previously injected into the culture vessel in which the substrate is installed. The number of liquid feeding ports to be arranged may be one per substrate, or two or more. 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 continuously or intermittently feed the liquid. Also, it may be automated or manual. After the liquid is once sent, the liquid existing 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 feeding and sucking a small amount of liquid 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 flow rate of the liquid sent in defoaming can be, for example, 100 cm/s or more. Also, it may be 200 cm/s or more, 250 cm/s or more, or 300 cm/s or more. Moreover, the upper limit includes a flow velocity at which the non-cell-adhesive substance on the substrate is not peeled off and the substrate is not damaged, for example, 2000 cm/s.

Also, the flow rate of the liquid for defoaming is not particularly limited, and is exemplified by 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, or 0.07 mL/s or more, and the upper limit is, for example, 20 mL/s.

In addition, the liquid transfer in the defoaming can be performed in an aseptic environment. In the present invention, as long as the recesses can be filled with a liquid, the cell culture substrate is subjected to known treatments required for cell culture (for example, exchanging the culture solution in a safety cabinet, leaving it to stand still in an incubator), if necessary. may

In this way, it is possible to prepare a cell culture vessel filled with a culture solution in such a manner that at least air bubbles are less likely to form or remain in the recesses. It should be noted that, by the above-mentioned liquid feeding, it is possible to remove air bubbles formed by filling the liquid in a place where no liquid exists or air bubbles formed after leaving the liquid for a while after filling.

Cultivation of cells and/or aggregates thereof in the obtained cell culture vessel cannot be generally set depending on the cells and/or aggregates thereof used, and can be appropriately carried out according to known techniques. The culture solution and culture conditions are not particularly limited. After culturing for a desired period of time, it can be subjected to the next collection.

In the present invention, liquid feeding is performed to collect cells and/or aggregates thereof. A device or instrument used for liquid transfer in recovery has, for example, at least a liquid transfer port with a diameter of 3 mm or less. Examples of the liquid feeding port include those having a liquid feeding port with a diameter of 3 mm or less, and may have a diameter of 2.5 mm or less, 2.0 mm or less, or 1.7 mm or less, for example. In addition, from the viewpoint of the liquid feeding flow rate and liquid feeding flow rate for exfoliating and / or releasing cells or aggregates thereof from the recess, the ratio of the diameter of the liquid feeding port to the diameter of the opening of the recess (diameter of the liquid feeding port /diameter of opening of recess) is preferably 30 or less, more preferably 20 or less, and even more preferably 10 or less. The lower limit is not particularly limited, and may be, for example, 0.1 or more. In addition, the diameter of the liquid feeding port means the maximum inner diameter of the liquid feeding port.

The shape and material of the liquid feed port are not particularly limited as long as it has a diameter of the size described above. For example, it is possible to use those manufactured or adjusted to have the above-mentioned bore in the liquid delivery port of a known dispenser, injection needles, blunt needle cannulas, tips, sondes, etc. having the above-mentioned bore.

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

The liquid for liquid transfer in recovery is not particularly limited. A physiological saline solution, a buffer solution, or the like may be used, but the culture solution itself may be used, or the culture solution previously injected into the cell culture vessel may be used.

The method of sending the liquid in the recovery may be such that the cells and/or aggregates in the recesses are exfoliated from the recesses by flowing out from the liquid delivery port, or released out of the recesses. Not limited. Specifically, for example, there is a method in which the tip of the liquid feed port is placed 0.1 to 10 mm above the upper end surface of the opening of the recess to feed the liquid. At that time, the tip of the liquid feeding port may be arranged so as to be immersed in the liquid previously injected into the culture vessel in which the substrate is installed. The number of liquid feeding ports to be arranged may be one per substrate, or two or more. 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 continuously or intermittently feed the liquid. Also, it may be automated or manual.

There is no particular limitation on the liquid feeding flow rate in the recovery, but it is preferable to feed the liquid at a predetermined flow rate. For example, 0.001 mL/s or more per 1 cm ² of opening area is exemplified. It may be 0.01 mL/s or more, 0.03 mL/s or more, 0.05 mL/s or more, or 0.07 mL/s or more, and the upper limit is, for example, 20 mL/s.

Also, the flow rate of the liquid sent in recovery is not particularly limited, and for example, 100 cm/s or more can be exemplified. Also, it may be 200 cm/s or more, 250 cm/s or more, or 300 cm/s or more. The upper limit includes a flow velocity at which the cell non-adhesive substance on the substrate is not peeled off and the substrate is not damaged, for example, 2000 cm/s.

In addition, the liquid transfer in the recovery can be performed under a sterile environment. In the present invention, if the cells and/or aggregates thereof can be exfoliated and/or released from the recessed portions, the cells and/or aggregates are present on the substrate in addition to the culture medium and the liquid-transferring liquid. Since they are present, for example, by collecting the liquid present on the substrate, the cells and/or aggregates themselves can be collected. There are no particular restrictions on the means used to collect the liquid present on the substrate, as long as it does not apply a strong force that damages the cells and/or aggregates thereof. For example, it may be collected by aspiration, or may be collected by pouring from the culture vessel. If necessary, additional liquid feeding may be performed to detach and/or release cells and/or aggregates thereof from the recesses. Collected cells and/or aggregates thereof can be subjected to known treatments (eg, passaging, freezing, fixing, sectioning, flow cytometric evaluation, microscopic observation, genetic analysis, transplantation).

In this manner, cells and/or aggregates thereof can be collected by feeding the liquid to the cell culture substrate. By such a method, cells and/or aggregates thereof can be easily collected and can be easily collected, so that they can be collected with high efficiency. In addition, since the collected cells and/or aggregates thereof are likely to be less damaged, the viable cell rate in the collected cell population is as high as 80% or more, for example. Therefore, the present invention can also provide a cell population with a viable cell rate of 80% or more, which is recovered by the above method.

EXAMPLES The present invention will be described in detail below with reference to Examples, but the present invention is not limited to these. In the following examples, room temperature means 20 to 30°C.

Production example 1
<Preparation of layer having cell adhesive surface (preparation of fluorine-containing polyimide film)>
25.332 g (0.057 mol) of 4,4′-hexafluoroisopropylidene diphthalic anhydride and 166.60 g of N-methylpyrrolidone were placed in a 500 mL three-necked flask 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 a nitrogen atmosphere, and held for 5 days. By doing so, a fluorine-containing polyamic acid resin composition (solid concentration: 15% by mass, 6FDA/TPEQ polyamic acid) was obtained. The polyamic acid had a weight average molecular weight of 153,000 and a viscosity of 6.7 Pa·s. The weight average molecular weight of the polyamic acid and the weight average molecular weight of the fluorine-containing polyimide after baking 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 baking was 40 μm to form a coating film. Then, the coating film was baked at 360° C. for 1 hour in a nitrogen atmosphere. Thereafter, the baked product was separated 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 sliding angle of 26.4°.

The methods for measuring physical properties in the above are as follows.
(Measurement of weight average molecular weight)
Apparatus: HCL-8220GPC manufactured by Tosoh Corporation
Column: TSKgel Super AWM-H
Eluent (LiBr.HO, 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 using polystyrene.
(Measurement of viscosity)
Device: Viscometer VISCOMETER TV-22 made by AS ONE
Setting: VI RANGE: H ROTOR No.6 SPEED: 10rpm
Standard liquid for viscometer calibration: Nippon Grease Co., Ltd. JS 14000
Measurement method: After calibrating with a standard solution for calibrating a viscometer, measure using 0.3 g of varnish. (Measurement temperature: 23°C)
(Measurement of static water contact angle)
Device: Automatic contact angle meter (manufactured by Kyowa Interface Science: DM-500)
Measurement method: Surface (cell non-adhesive surface or cell adhesive surface) or film (film formed of a substance exhibiting cell non-adhesive property or cell adhesive property) immediately after 2 µL of water is dropped on the droplet adhesion angle Measure (measurement temperature: 25°C).
(Measurement of falling angle)
Device: Automatic contact angle meter (manufactured by Kyowa Interface Science: DM-500)
Measurement method: After dropping 25 μL of water on a surface (non-adhesive cell surface or cell-adhesive surface) or film (film formed of a substance exhibiting cell non-adhesiveness or cell adhesiveness), the sheet is continuously tilted. The angle at which the liquid falls is taken as the falling angle (measured temperature: 25°C).

<Preparation of layer having cell non-adhesive surface>
A transparent PET film (thickness: 250 μm) is attached to a transparent PET film (thickness: 250 μm) after peeling off the peeling tape on one side of the double _-sided tape (thickness: 25 μm). (Formed through-holes: 400/cm ² , 42000/sheet, laser incident side hole diameter 500 μm, laser emission side hole diameter 300 μm). After that, using a spin coater (Mikasa: MS-A150) on the surface of the PET film side, MPC polymer solution (0.5% ethanol solution) was coated to a thickness of 0.05 μm, and dried in a dryer at 50 ° C for 2 hours. A layer with a non-adherent surface to cells [static water contact angle of PET film coating layer (MPC polymer coating layer) side: 107.5°] was obtained by drying for a period of time.

<Preparation of cell culture sheet/culture vessel>
Next, the layer having a cell-adhesive surface prepared above was attached to the surface of the layer having a cell-non-adhesive surface on the side of the double-sided tape from which the other peeling tape was removed, to prepare a cell culture sheet. (Sheet thickness: 315 μm). The resulting cell culture sheet was placed in a culture plate to complete a container used for cell culture.

Example 1
<Expansion culture of cells>
Human adipose derived stem cells (AdSC) were used as cells. AdSC was purchased from a manufacturer (Lonza, PT-5006) and used.

Frozen cells were thawed in a constant temperature water bath at 37° C. and added to 9 mL of KBM ADSC-2 medium (basal medium, 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 basal medium in advance so that the total volume after adding the cell suspension ^is 30 mL in an 800 mL cell culture flask with a filter cap (manufactured by Sumitomo Bakelite). / flask, and cultured (expansion culture) in a 5% (v/v) CO ₂ incubator at 37°C.

<Degassing treatment>
Separately, the culture vessel prepared in Production Example 1 was defoamed. Specifically, about 20 mL of PBS was first added to the culture vessel in a safety cabinet. Next, fill 20 mL of PBS into a 20 mL all-plastic (Nipro) with an 18G non-beveled needle (inner diameter: 0.9 mm, manufactured by Terumo), and place the tip of the needle in the PBS liquid inside the container (above the concave opening). Approximately 2 mm above the end surface), push out the syringe while moving it horizontally to transfer the entire amount of the liquid to about 1/3 of the entire bottom surface of the culture vessel in about 8 seconds. After sucking up and refilling the syringe with PBS, the same liquid feeding was repeated for the remaining area to perform defoaming. Air bubbles were no longer observed in most of the wells, but the above operation was performed in the same way for the portions where air bubbles remained. Then, after standing for 15 minutes in a 5% (v/v) CO ₂ incubator at 37°C, the PBS filled in the syringe was extruded again to remove newly generated air bubbles. After such degassing, remove the PBS so that it does not completely disappear from the recess, then add 21 mL of KBM ADSC-2 medium containing 1% antibiotics and add 5% (v/ v) It was allowed to stand overnight in a CO ₂ incubator to obtain a defoamed culture vessel.

<Preparation of spheroids>
After removing the medium from the culture flask and adding 5 mL of cell detachment solution accutase (manufactured by Promocell), the cells were detached by holding in a 5% (v/v) CO ₂ incubator at 37°C for about 5 minutes. Then, the stripping solution was collected and transferred to a tube using PBS so that the total volume was 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. After that, the concentration was adjusted to 1.0×10 ⁶ cells/mL.

The medium was removed from the culture vessel which had been defoamed by allowing it to stand overnight in a 5% (v/v) CO ₂ incubator at 37°C, and cells were seeded at 500 cells/well. After standing for 15 minutes in a safety cabinet, the cells were placed in a 5% (v/v) CO ₂ incubator at 37°C and cultured for 3 days.

<Collection of spheroids>
The culture medium of the vessel cultured for 3 days was removed by aspiration and 20 mL of PBS was added. Next, fill 20 mL of PBS into a 20 mL all-plastic (Nipro) with an 18G non-beveled needle (inner diameter: 0.9 mm, manufactured by Terumo), and place the tip of the needle in the PBS liquid inside the container (above the concave opening). Approximately 2 mm above the end surface), the syringe is pushed out while moving horizontally, and the liquid is delivered to 1/3 of the entire concave portion of the container in about 8 seconds (approximately 0.09 mL/s per 1 cm ² of opening area, flow rate of approximately 393 cm/s). ) was performed to peel the spheroids from the substrate. The exfoliated spheroids and PBS were sucked up with 20 mL all plastic and collected in a 50 mL centrifuge tube.

<Calculation of spheroid recovery efficiency>
The container from which the spheroids were peeled and collected was photographed with a plate scanner (manufactured by Screen), the obtained image was divided into 9 parts, the spheroid collection efficiency at each point was calculated by the following formula, and the average value was obtained.
Recovery efficiency (%) = {(number of spheroids present on plate before recovery) - (number of spheroids present on plate after recovery)} / (number of spheroids present on plate before recovery) x 100

In addition, the viability of cells in the collected spheroids was measured as follows. The spheroid dispersion was centrifuged at 210×g for 5 minutes, suspended in 7 mL of Accumax (manufactured by Nacalai Tesque), and treated at room temperature for 30 minutes. The cell suspension filtered through a cell strainer (manufactured by Corning) was centrifuged at 210×g for 5 minutes, suspended in 1 mL of PBS, and the viability was calculated by counting the number of cells.

<Results>
By using a syringe, spheroids could be collected without PBS leaking out of the container. The recovery efficiency was 93±3% (average of 9 sites). Spheroids could be recovered without removing the container from the sterile environment and without using a large-sized device (see FIGS. 1 and 2).

In addition, the recovered spheroids had a high viable cell rate of 98%, indicating that the recovery method causes less damage to the cells.

INDUSTRIAL APPLICABILITY According to the method of the present invention, damage can be easily reduced, and cells and/or aggregates thereof can be collected easily and efficiently. be able to.

Claims (10)

A liquid-feeding solution is sent from a liquid-feeding port with a diameter of 3 mm or less at a flow rate of 100 cm/s or more to a cell culture substrate containing cells in a concave portion with a diameter of 1000 μm or less. A method for recovering cells and/or aggregates thereof, comprising a step of liquefying, wherein the cell culture substrate has a plurality of recesses having openings with a pore size of 1000 μm or less in diameter, and the inner surfaces of the recesses are non-adherent to cells. a cell culture sheet having an adhesive surface, and the bottom surface of the recess has a cell-adhesive surface . The method according to claim 1, wherein the liquid is fed at a flow rate of 0.001 mL/s or more per 1 cm ² of opening area. 3. The method according to claim 1, wherein the liquid is sent from 0.1 to 10 mm above the recess. 4. The method according to any one of claims 1 to 3, wherein the diameter of the liquid feed port is 2.5 mm or less. 5. The method according to any one of claims 1 to 4, wherein the ratio of the diameter of the liquid feed port to the diameter of the opening of the recess (diameter of the liquid feed port/diameter of the opening of the recess) is 30 or less. The substrate for cell culture has a plurality of recesses with openings having a pore size of 1000 μm or less in diameter, the inner surfaces of the recesses having cell non-adhesive surfaces, and the bottom surfaces of the recesses having cell adhesive surfaces. The method according to any one of claims 1 to 5, which is a cell culture sheet. The method according to any one of claims 1 to 6, which is carried out in an aseptic environment. The method according to any one of claims 1 to 7, further comprising a step of recovering liquid present on the substrate after the liquid transfer step. The method according to any one of claims 1 to 8, wherein the cells form spheroids. The method according to any one of claims 1 to 9 , wherein the cells and/or aggregates thereof are cell populations with a viable cell rate of 80% or more .

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