JP7421909B2 - How to transport cells - Google Patents

Description

The present invention relates to a novel method for transporting cells, a container for transporting cells, and the like.

In cell culture, techniques for three-dimensionally culturing cultured cells have recently been attracting attention from the viewpoint of being able to more closely imitate living tissue.

For example, Patent Document 1 describes a microchip having a plurality of cell retention cavities, in which the bottom surface of the cavity has an adhesive region exhibiting cell adhesive properties, and a non-adhesive region surrounding the adhesive region exhibiting cell non-adhesive properties. It is described that by including the above, a cell tissue body having a uniform shape and size can be formed. Further, Patent Document 2 discloses a method of culturing cells on a porous film having through holes, in which the surface of the porous film is composed of a cell adhesive region and a cell non-adhesive region, and A method for efficiently culturing a three-dimensional tissue while smoothly supplying a culture solution and discharging waste products has been disclosed.

Japanese Patent Application Publication No. 2006-121991 Japanese Patent Application Publication No. 2008-199962

An object of the present invention is to provide a novel method for transporting cells.

Another object of the present invention is to provide a method for transporting cells that can be transported while suppressing cell collapse.

Another object of the present invention is to provide a method for transporting cells that can be transported while suppressing cell aggregation.

Another object of the present invention is to provide a method for transporting cells that can be transported while suppressing a decline in cell function.

Since cells obtained by culture may be damaged if removed from the cell culture container, it is considered ideal to transport the cells without removing them. However, in reality, if cells are transported in a container after culturing, the cells may flow out of the container or collide with the inner surface of the container, so such a method is not practical. Normally, cells were removed from the cell culture container and transported in a transport container.

Under these circumstances, the present inventors came up with the idea of transporting cells by adhering them to a cell culture container.
However, even if cells are adhered to and cultured in a cell culture container, if the cultured cells are transported while still in the container, some of the cells may collapse or the cells may aggregate and become large. It has been difficult to transport cells while adhering them to a cell culture container due to problems such as oxidation.

Under these circumstances, the present inventors surprisingly found that when cells are transported by holding (or contacting or adhering) them to a substrate having a cell-retaining surface containing a polyimide resin, the cells and the substrate can be transported. It was found that cell disintegration and aggregation can be suppressed, probably due to the moderate adhesiveness of the material.
Furthermore, it has been found that even if cells obtained by culturing on the cell-retaining surface of the base material are transported while being retained on the base material, cell collapse and aggregation can be suppressed.

The present inventors have obtained various new findings in addition to the above, and have completed the present invention after further intensive studies.

That is, the present invention relates to the following inventions, etc.
[1]
A method for transporting cells by holding and transporting cells on a base material having a cell-retaining surface containing polyimide resin.
[2]
The method according to [1] above, wherein the cell-retaining surface has a cell-adhesive site, and the cell-adhesive site has a static water contact angle of 70° or more.
[3]
The method according to [1] or [2] above, wherein the polyimide resin is a fluorine-containing polyimide resin.
[4]
The method according to any one of [1] to [3] above, wherein the cells to be retained on the substrate are cells cultured at a cell adhesive site of the substrate.
[5]
The method according to any one of [1] to [4] above, wherein the cell adhesion site of the substrate is divided by a wall, and the wall has a cell non-adhesion site.
[6]
The base material 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 region, and the bottom surface of the recess has a cell adhesive region containing polyimide resin. The method according to any one of [1] to [5] above, which is a base material having.
[7]
The method according to any one of [1] to [6] above, wherein the cells are transported while forming spheroids.
[8]
The method according to any one of [1] to [7] above, wherein the cells are transported while maintaining the culture environment.
[9]
The method according to any one of [1] to [8] above, which is transported at a temperature of 27 to 37°C.
[10]
A defoaming step that includes a step of sending a liquid feeding liquid from a liquid feeding port with a diameter of 3 mm or less and/or at a flow rate of 100 cm/s or more, and/or a defoaming process that includes a step of feeding a liquid feeding liquid at a flow rate of 100 cm/s or more; The method according to any one of [1] to [9] above, comprising a recovery step that includes a step of sending the liquid feeding liquid from the following liquid feeding port and/or at a flow rate of 100 cm/s or more. .
[11]
A container for transporting cells, comprising at least a base material having a cell-retaining surface containing a polyimide resin.

The present invention can provide a novel method for transporting cells.

Further, according to the transport method of the present invention, cells can be transported while suppressing collapse (or generation of damage) and aggregation due to transport. In particular, cell aggregates (spheroids) can also be transported while forming spheroids. According to the transport method of the present invention, it is possible to suppress the collapse and aggregation of cells due to transport, so that the cells become dead cells due to disintegration, the cells cannot be used for the intended purpose, and the cells become gigantic. It can prevent oxygen and nutrients from reaching the center of cells, leading to necrosis, and a decline in cell function.

Furthermore, according to the transport method of the present invention, the substrate holding the cells after culture can be transported as is, so the cell culture environment (e.g., temperature, CO ₂ concentration, pH of the medium, etc.) can be maintained ( or without significant changes). Therefore, according to the transport method of the present invention, it is possible to suppress the decline in cell function caused by transport.
In addition, in the present invention, it is assumed that by holding and transporting cells on a substrate having a cell-retaining surface containing polyimide resin, cellular factors will be expressed and signals that maintain cell survival and function will enter the cells. be done. Furthermore, it is presumed that the present invention is also useful in transport, perhaps in connection with the introduction of signals in this way.

FIG. 1 is a diagram schematically showing the cross-sectional structure of one embodiment of the base material used in the present invention. FIG. 2 is a diagram schematically showing the cross-sectional structure of one embodiment of the base material used in the present invention. FIG. 3 is a diagram schematically showing the cross-sectional structure of one embodiment of the base material used in the present invention. FIG. 4 is a diagram schematically showing an example of a fine pattern for one embodiment of the base material used in the present invention. FIG. 5 is a photomicrograph of the culture container before and after transportation in Example 1.

The cell transport method of the present invention is a method of transporting cells while holding them on a specific substrate described below.

<Base material>
The base material has a cell-holding surface (or a cell-holding portion, that is, a surface on which cells are held, or a side on which cells are held). The present invention is characterized in that a base material containing polyimide resin is used for this cell-retaining surface.

The substrate usually has at least a cell adhesive site in order to retain (or adhere to, or even transport) cells. Although the relationship (positional relationship) between such a cell adhesive site and the polyimide resin is not limited, typically, the cell adhesive site contains a polyimide resin [or is formed of a polyimide resin]. )] in many cases.

That is, typical substrates include substrates that have a cell-adhesive site containing polyimide resin on the cell-retaining surface.

In the cell retention surface, the cell adhesion site can be a cell culture surface.
Moreover, the cell-retaining surface may have a cell non-adhesive area.
Note that the shape of the base material is not particularly limited, and may be, for example, a sheet (or film) or the like. For example, the base material may be a sheet having a cell-retaining surface (one surface is a cell-retaining surface and the other surface is a non-cell-retaining surface).

(Cell adhesive site)
The cell adhesion site may be, for example, a site to which cells adhere at certain adhesion points. Alternatively, it may be a site to which cells adhere so as to be immobilized to the extent that they can be peeled off by liquid flow such as pipetting. It may also be a site where cells adhere and are maintained or proliferated two-dimensionally, or a site where cells can form a three-dimensional or three-dimensional tissue such as a layer or a spheroid. It may be a part to be bonded.

Examples of polyimide resins include polyimide resins containing structural units represented by the following formula (I).
Further, from the viewpoint of cell adhesion (and transportability) and culturability, 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 acid.
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.

Examples of 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 a polyimide consisting of:

Examples of the resin having a fluorine atom include 4,4'-hexafluoroisopropylidene diphthalic anhydride (6FDA)/1,4-bis(aminophenoxy)benzene (TPEQ) copolymer, 6FDA/4,4 '-Oxydiphthalic anhydride (ODPA)/TPEQ copolymer, 4,4'-(4,4'-isopropylidenediphenoxy)diphthalic acid (BPADA)/2,2-bis[4-(4-aminophenoxy) ) phenyl]hexafluoropropane (HFBAPP), 6FDA/2,2-bis(4-(4-aminophenoxy)phenyl)propane (BAPP) copolymer, 6FDA/2,2'-bis(trifluoromethyl)benzidine (TFMB) copolymer, 6FDA/4,4'-diaminodiphenyl ether (ODA) copolymer, 6FDA/4,4'-bis(4-aminophenoxy)biphenyl (BAPB) copolymer, etc. with the following formula ( Examples include fluorine-containing polyimide resins containing the structural unit represented by I); ethylene-tetrafluoroethylene copolymers, and the like.

In the above formula (I), X ⁰ represents either an oxygen atom, a sulfur atom, 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 alkylene group, arylene group, aryleneoxy group, arylenthio group, etc. is 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 cell adhesiveness and culturability, the divalent organic group represented by X ⁰ is preferably selected from the group consisting of b-2 to b-10 and c-2 to c-10. Preferably, the structure is selected from the group consisting of b-7 to b-9 and c-7 to c-9, and the structure represented by b-8 is even more preferable. Further, it is equally preferable that the divalent organic group represented by X ⁰ is -C(CF ₃ ) ₂ -.

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 cell adhesion and culturability, in the above formula (I), Y is 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 cell adhesion and culturability, 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 selected from the group consisting of b-2 to b-10 and c-2 to c-10; and Y is d-3, d-9, e-1 to e-4, f-6, and f- selected from the group consisting of 7. 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 is preferably 5.0% in view of the polymerization reactivity and viscosity of the resulting resin composition after polymerization, and ease of handling during subsequent film formation and baking. It is at least 10% by weight, more preferably at least 10% by weight, preferably at most 50% by weight, and even more preferably at most 40% by weight.

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 composition containing a 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. , more preferably 100 to 380°C, preferably 0.1 to 10 hours, more preferably 0.2 to 5 hours, to perform an imidization reaction to obtain a resin composition containing polyimide. be able to.

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. There may be only one type of tertiary amine, or two or more types may be used.

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 is a value measured by the following method. When the weight average molecular weight is within the above range, polyimide resin synthesis and handling, film formation, cell adhesion, cell culturability, etc. will be better.

(Measurement of weight average molecular weight)
Equipment: HCL-8220GPC manufactured by Tosoh Corporation
Column: TSKgel Super AWM-H
Eluent (LiBr・H ₂ O, 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.

The cell adhesion site may be made of polyimide resin only, and may not contain other substances other than polyimide resin [e.g., substances exhibiting cell adhesion, additives (e.g., plasticizers, antioxidants, etc.)]. It's okay to stay.
Substances exhibiting cell adhesive properties are not particularly limited and can be used as long as the cells to be used adhere to them or can bind to cell surface molecules such as proteins and sugar chains present in the cell membrane of the cells to be used. . It may be either hydrophilic or hydrophobic, but from the viewpoint of cell adhesion and culturability, hydrophilic (especially hydrophilic but not superhydrophilic) or hydrophobic ( In particular, those that are hydrophobic (not superhydrophobic) are preferred, and those that exhibit hydrophobicity are more preferred. Further, the degree of adhesiveness of the substance exhibiting cell adhesiveness may be such that cells do not jump out from the recesses when using the substrate 1 described below. Examples of such substances include substances obtained from living organisms or synthesized, such as proteins (collagen, fibronectin, laminin, etc.) and synthetic resins other than polyimide resins (fluororesin, polysulfone, polyether, etc.). sulfone, polydimethylsiloxane, mixtures thereof, etc.).

In addition, even if the cell-adhesive site contains a substance that exhibits cell-adhesive properties, it must mainly contain polyimide resin (for example, the content ratio of polyimide resin is 50% of the total amount of polyimide resin and the substance that exhibits cell-adhesive properties). % by mass or more) is preferable.

From the viewpoint of cell adhesion and culturability, the static water contact angle of the cell adhesion site may preferably be 70° or more, more preferably 75° or more (for example, more than 75°), and Preferably it is 77° or more, more preferably 79° or more, even more preferably 80° or more (for example, more than 80°).
Further, the upper limit of the static water contact angle of the cell adhesion site is, for example, less than 150°, preferably 120° or less (for example, less than 120°), more preferably 110° or less, and still more preferably 100° or less ( For example, the angle may be less than 99°, 98° or less, 97° or less, 95° or less, etc.).

Note that the static water contact angle of the cell adhesion site may be in an appropriate range (for example, 70° or more and 120° or less) by appropriately combining these upper and lower limits.

The falling angle of the cell adhesion site is preferably 15° or more, 18° or more, 19° or more, 20° or more, 22° or more, 24° or more, 26° or more, from the viewpoint of cell adhesion and culturability. The higher the angle is, the more preferable it is, in the order of 1° or more, 28° or more, and 30° or more.
Further, the upper limit of the falling angle is, for example, less than 80°, preferably 70° or less (e.g., less than 70°), more preferably 60° or less (e.g., less than 60°), and even more preferably It may be 50° or less (for example, less than 50°).
Note that the falling angle of the cell adhesion site may be within an appropriate range (for example, 15° or more and less than 80°) by appropriately combining these upper and lower limits.

Further, the cell adhesive site may have a static water contact angle of 70° or more and a falling angle of 15° or more. The above static water contact angle and falling angle may be values measured by the following method.

(Method of measuring static water contact angle)
Equipment: Automatic contact angle meter (Kyowa Interface Science: DM-500)
Measurement method: Measure the adhesion angle of the droplet immediately after dropping 2 μL of water onto the surface (non-cell-adhesive surface or cell-adhesive surface) or film (film formed from a non-cell-adhesive or cell-adhesive substance). (Measurement temperature: 25°C).

(Measurement method 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 cell non-adhesive or cell adhesive material), the substrate is continuously tilted. The angle at which it flows down is defined as the falling angle (measurement temperature: 25°C).

(Cell non-adhesive site)
A cell non-adhesive site is, for example, 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 weakly. Even if it does, it may be a site that naturally detaches.

The cell non-adhesive site may be formed of a substance that exhibits at least cell non-adhesive properties.
The substance exhibiting cell non-adhesion properties is not particularly limited and can be used as long as it does not adhere to the cells used 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. Further, the material may be either hydrophobic or hydrophilic; for example, it may be superhydrophobic (superhydrophobic) or superhydrophilic. From the viewpoint of cell non-adhesion, uniformity and formability of cells obtained by culturing, hydrophobicity (especially superhydrophobicity) [e.g., cell adhesive site or resin constituting the site (and its contact angle) ) is particularly preferable; ) is also preferable.

Substances exhibiting cell non-adhesive properties include, for example, compounds containing ethylene glycol and its derivatives, MPC (2-methacryloyloxyethylphosphorylcholine) and its derivatives, HEMA (hydroxyethyl methacrylate) and its derivatives, or polymers of these compounds. , compounds containing SPC (segmented polyurethane) and its derivatives, proteins obtained from living organisms (albumin, etc.), and sugar chains to which cells do not adhere (agarose, cellulose, etc.) are selected as appropriate depending on the cell type. It can be used as These substances can be used alone or in combination of two or more.
Among these, MPC, derivatives thereof, or polymers thereof are preferred from the viewpoints of adhesion to cell adhesion sites, simplification of the manufacturing process of the substrate, improvement of uniformity of cells obtained by culturing, and the like.
Note that the substance may be appropriately modified depending on the handleability, the desired degree of hydrophobicity (for example, superhydrophobicity) and hydrophilicity (for example, superhydrophilicity), and the like. 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 appropriately hydrophobized or hydrophilized (e.g., introduction of a hydrophobic group or hydrophilic group, etc.) to obtain the desired hydrophobic or hydrophilic property. You can also get the material.

The static water contact angle of the cell non-adhesive area is determined by the fact that the cell non-adhesive area is hydrophobic from the viewpoint of cell non-adhesion, uniform cell size obtained by culture, and improving circularity. In this case, the angle may be 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, when the cell non-adhesive site is hydrophilic, the static water contact angle may be preferably 65° or less, more preferably 55° or less, and still more preferably 50° or less. Further, the angle may be 0° or more, preferably 5° or more, and more preferably 10° or more.
For example, in a highly hydrophobic MPC (or a site formed by the MPC), a static water contact angle of, for example, 90° or more or 100° or more can be achieved. .
Note that such a static water contact angle may be a value at a cell non-adhesive site, or a value at a substance exhibiting cell non-adhesive property (or a substance constituting a cell non-adhesive site). good.
Further, the static water contact angle may be measured, for example, by the method described above.

In addition, it is important to balance the degree of adhesion between cell-adhesive sites and non-adhesive sites in terms of improving cell transportability and the size uniformity and circularity of cells obtained by culture. be. Therefore, even if the cell non-adhesive site exhibits adhesiveness, it is sufficient that the adhesiveness is lower than that of the cell-adhesive site. For example, when using the above static water contact angle as an index, the difference (or its absolute value) between the static water contact angles between the cell adhesive site and the cell non-adhesive site is 3° or more (for example, 5° or more). The angle is preferably 10° or more (for example, 12° or more), and even more preferably 15° or more. Further, the upper limit can be appropriately selected depending on the combination of hydrophobicity and hydrophilicity of the cell non-adhesive site and the cell adhesive site, and is not particularly limited, but for example, 100°, 90°, 80°, 70°, It may be 60°, 50°, 40°, 30°, etc.

(Structure of base material)
The substrate is not particularly limited as long as it can hold (and even transport) cells. For example, in the base material, the aspect (e.g., position, form) of the cell adhesive site (cell adhesive site present on the cell-retaining surface) is not particularly limited, and The surface) may be formed (constituted) of only a cell-adhesive site, or may be formed (constituted) of a cell-adhesive site and a cell-non-adhesive site.

Further, in the base material (cell-retaining surface), there may be a plurality of cell-adhesive sites (eg, partitioned or compartmentalized).
In such a base material, the cell adhesive site (multiple cell adhesive sites) may be partitioned by a wall (for example, may constitute part or all of the recess), and the cell adhesive site (multiple cell adhesive sites) may be partitioned by a wall (for example, may constitute part or all of the recess), The areas may be divided into sections, or these may be combined.
For example, a cell adhesive site in the base material may be divided by a cell non-adhesive wall (or a wall having a cell non-adhesive site). Typically, such a base material may be a base material having a plurality of concave portions whose bottom surface (bottom) is a cell adhesive site and whose wall surface (inner surface) is a cell non-adhesive site.

Further, in the base material, the polyimide resin may be contained in the cell-retaining surface, but as described above, it is typically contained in the cell-adhesive site. In such a case, the polyimide resin only needs to be included at least in the cell adhesion site [particularly its surface (part that comes into contact with cells)], and may constitute (form) part or all of the cell adhesion site. You can.

The same applies when the base material has a cell non-adhesive area, and the cell non-adhesive substance only needs to be contained at least in the cell non-adhesive area (especially the surface thereof), and it is sufficient that the cell non-adhesive substance is contained at least in the cell non-adhesive area (particularly on the surface thereof). Alternatively, all of them may be configured (formed).

Below, typical base materials will be explained in detail.
One embodiment of the substrate in which the cell adhesion site is partitioned includes, for example, a plurality of recesses each having an opening diameter of 1000 μm or less, and the inner surface of the recess has a cell non-adhesion site; Further, a base material (hereinafter sometimes simply referred to as "base material 1") in which the bottom surface of the recess has a cell adhesion site containing a polyimide resin can be mentioned.
The structure of the base material 1 will be explained using an example of a cross-sectional view of the base material (sheet) cut in a direction perpendicular to the recess. As shown in the schematic diagram of FIG. 1, a plurality of recesses 11 are present on the sheet surface 12, and the recess 11 is composed of an inner surface 11a and a bottom surface 11b, and cells are held and cultured within the recess 11.
Moreover, in FIG. 1, the inner surface 11a has a cell non-adhesive region, and the bottom surface 11b has a cell adhesive region.

The number of recesses cannot be set unconditionally depending on the sheet area, the type of cells to be cultured, etc., but can be set as appropriate according to the common technical knowledge. For example, the lower limit number per unit area (cm ² ) may be 1 piece, but examples include 10 pieces, 20 pieces, 30 pieces, 50 pieces, etc., and the upper limit number is 1000 pieces, 500 pieces, 300 pieces, 200 pieces. , 100, etc. Further, the total number of recesses on the sheet surface can be set as appropriate, for example, 10 or more, 100 or more, 1000 or more, 10000 or more, 50000 or more.

The shape of the opening of the recess is not limited to a circle, and may be, for example, a polygon or an ellipse.
The diameter of the opening is preferably 1000 μm or less in diameter, and can be appropriately set according to the common general knowledge in the art depending on the size of the cells to be cultured. In the present invention, the aperture refers to the diameter (maximum length) of a circle formed so as to enclose the target part, regardless of the shape of the target part, and the aperture of the opening is, for example, 1, it is the length indicated by D(11). Examples of the diameter of the opening are in the range of 10 to 1000 μm, 10 to 700 μm, 10 to 600 μm, and 10 to 500 μm.

The shape of the bottom of the recess is not limited to a circle, and may be, for example, a polygon or an ellipse, and may be the same or different from the shape of the opening. Also, 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 aperture of the bottom surface is, for example, the length indicated by DB (11b) in FIG. Examples include ranges of 10 to 400 μm and 10 to 300 μm. For example, 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 diameter to the opening diameter (bottom diameter/opening diameter) is not particularly limited, but from the viewpoint of ease of cell seeding and collection, it is 5/1 to 1/2. Examples include 5, 3/1 to 1/3, and 1/1 to 1/2.

Further, the distance (gap) between an opening and an adjacent opening is, for example, the length indicated by D (12) in FIG. Depending on the cell culture, the range may be, for example, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 300 μm or less, 200 μm or less, 100 μm or less, and any finite value may be used.

The depth of the recess can be appropriately set according to the size of the cells to be cultured according to the common general knowledge in the art. The depth of the recess is, for example, the length indicated by D (11a) in FIG. 1, and is, for example, within the range of 10 to 300 μm or 10 to 1000 μm.

The ratio of the opening diameter to the depth of the recess (opening diameter/recess depth) is not particularly limited, but from the viewpoint of ease of cell seeding and collection, it is 5/1 to 5/1. Examples include 1/5, 3/1 to 1/3, and 2/1 to 1/2. When it is within the above range, cells are less likely to jump out of the recesses, and operations such as cell collection and defoaming treatment become easier.

The thickness of the bottom surface of the recessed portion is not particularly limited, and can be appropriately set according to the common technical knowledge.

In the base material 1, the cell non-adhesive region may be formed by physically or chemically fixing a substance exhibiting cell non-adhesive property to the base material surface constituting the recess, and may be formed by physically or chemically fixing a substance exhibiting cell non-adhesive property to the base material itself. may be made of a substance that exhibits non-cell adhesive properties.
When a substance exhibiting cell non-adhesion is immobilized on the substrate surface, as shown in the schematic diagram of FIG. has been done.

In addition, the immobilization of substances exhibiting cell non-adhesive properties can be achieved by drying a solution containing them on the surface of the substrate, by melting and pressing the substance, or by exposing the substance applied to the substrate to UV or other light. A method of curing with energy rays, which causes 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 to form covalent bonds. It can be immobilized on the surface of the substrate by a method of forming a thiol group of the substance and a thin film of metal (platinum, gold, etc.) previously formed on the substrate. The thickness upon immobilization is not particularly limited, and is exemplified by 0.01 to 1000 μm.

The proportion of the inner surface of the recess occupied by the cell non-adhesive region is not particularly limited, but is preferably to an extent that reduces cell adhesion. Preferably, it occupies 90% or more of the area of the inner surface, more preferably 95% or more, particularly preferably all of it.

In addition, in the base material 1, the cell adhesive site is formed by physically or chemically applying a resin (i.e., a polyimide resin or a resin composition containing a polyimide resin) constituting the cell adhesive site to the base material surface constituting the bottom surface of the recess. The resin constituting the cell-adhesive site may be placed outside the recess, and the base material itself constitutes the cell-adhesive surface. It may also be made of resin.
When the base material itself is made of resin constituting the cell adhesion site, as shown in the schematic diagram of FIG. You can leave it there.

The base material 1 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, the base material 1 may be a laminate of a layer having a cell non-adhesive site having through-holes and a layer having a cell adhesive site.

The layer having a cell non-adhesive area may be a layer in which a substance exhibiting cell non-adhesion is immobilized on a layered base material, or a layer consisting of a substance exhibiting cell non-adhesion; From the viewpoint of forming pores or from the viewpoint of simplifying the manufacture of the substrate, it is preferable to use a layered substrate in which a substance exhibiting cell non-adhesion is immobilized. This configuration makes it easier to manufacture the base material, and furthermore, by forming through holes or recesses in the base material and then fixing a substance exhibiting cell non-adhesive properties, it is possible to form the base material in the recesses. It is also possible to prevent damage to non-cell adhesion sites during formation, which is preferable from the viewpoint of improving cell retention and culture characteristics.

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

The immobilization of the substance exhibiting cell non-adhesion to the layered base material can be carried out in the same manner as the method of immobilizing the substance exhibiting cell non-adhesion on the inner surface of the recesses described above. Note that, from the viewpoint of workability, it is preferable to perform immobilization after forming through holes, which will be described later.

The layer having a cell non-adhesive site preferably includes the surface of the base material and a through hole. The wall of the through hole preferably 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 site, but it also corresponds to the depth of the recess described above, 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, and more preferably 10 nm or more. The thickness of the entire layer including the layer of the substance exhibiting cell non-adhesive properties and the base material can be appropriately set as long as it falls within the range of the thickness of the layer having the above-mentioned cell non-adhesive site.

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. The processing may be such that the shape of the through hole becomes a tapered shape, and at that time, the periphery of the end portion is deformed, for example, the edge of the opening and the opening adjacent to the opening are deformed. A structure may be formed in which the layer thickness of the portion located in the middle region of the portion is different.

The layer having the cell adhesive site may be a layer in which a resin constituting the cell adhesive site is immobilized on a layered base material, or a layer made of the resin constituting the cell adhesive site.

The thickness of the layer having the cell adhesive site 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 thickness of the bottom surface of the recess described above.

The method for manufacturing the substrate 1 is not particularly limited, but from the viewpoint of manufacturing efficiency, for example, a layer having a cell non-adhesive site and a layer having a cell adhesive site having a plurality of through holes with a diameter of 1000 μm or less are laminated in this order. You may also use the method of

The above-mentioned method can be used to prepare each layer, ie, a layer having a cell non-adhesive site and a layer having a cell adhesive site. The same applies to the formation of through holes in a layer having a cell non-adhesive site.

The lamination method may be one in which pre-prepared layers are laminated in order, a method in which a layer is separately formed on a pre-prepared layer, or a combination thereof. Specifically, for example, a resin constituting the cell adhesion site is cast, spin coated, etc. on a release sheet whose surface has been subjected to release treatment (e.g., an organic polymer film such as a polyethylene base material, ceramics, metal, etc.). A layer having cell-adhesive sites can be formed into a sheet by applying it to an appropriate thickness using a method such as roll coating and heating it. On the other hand, after forming through-holes in the base material of the layer having cell non-adhesive regions, the surface is coated with a substance exhibiting cell non-adhesive properties to prepare a layer having cell non-adhesive regions in advance. Can be done. Then, after peeling off the release sheet of the layer having the cell-adhesive site molded above, it can be manufactured by laminating a separately prepared layer having the cell-non-adhesive site. In addition, when laminating a layer having a cell non-adhesive site and a layer having a cell adhesive site, the layer may be laminated using the adhesive layer (adhesive layer) described above, or by welding (high frequency welding, ultrasonic welding). etc.), or may be laminated by compression bonding (thermocompression bonding, etc.).

Another embodiment of the substrate in which the cell adhesion sites are divided is, for example, a substrate having a layer having the cell adhesion sites and a fine pattern of a cell non-adhesive substance provided on the surface of the layer. (That is, a base material having a mask of a fine pattern of a cell non-adhesive substance in a layer having a cell adhesive site) (hereinafter sometimes simply referred to as "base material 2").

The fine pattern includes a case where it is composed of a plurality of unit shapes and a case where it is composed of a shape corresponding to a gap between adjacent unit shapes, and is formed of a cell non-adhesive substance. For example, when the micropattern of the cell non-adhesive substance is configured in a shape corresponding to the gap between adjacent unit shapes, the cell adhesive site is exposed in the unit shapes. Further, an example of the base material 2 is schematically shown in FIG. 4 (3 is a unit shape, 4 is a gap), and 3 is a fine pattern of a cell non-adhesive substance, 4 is a cell adhesive site, and 3 An example is an embodiment in which 4 is a cell adhesive site and 4 is a fine pattern of a cell non-adhesive substance. Note that the unit shape here is also referred to as a unit pattern or unit type.

There are no particular limitations on the shape of the unit shape, and examples include those made up of straight lines, curved lines, surfaces, and combinations of these.

Specifically, examples include polygonal, elliptical, circular, fan-shaped, etc., and two or more of these may be combined to form one unit type. Polygons include hexagons, pentagons, quadrilaterals (including, for example, squares, rectangles, parallelograms, rhombuses, and trapezoids), triangles, and star shapes, and circular shapes include circles as well as approximately circular shapes. . The unit shape may be a plurality of unit shapes having the same size or different sizes as long as a fine pattern can be formed.

For example, in the case of a unit shape having a region (section), the average equivalent circular diameter (maximum diameter) of the unit shape may be 1000 μm or less, and may be 900 μm or less, or 800 μm or less. The lower limit can be appropriately set depending on the type of cells as long as the cells can be retained. For example, 30 μm may be mentioned. Further, in order to prevent adjacent cells from meeting each other, the center-to-center distance between perfect circles corresponding to adjacent unit shapes may be, for example, 1500 μm or less, preferably 1000 μm or less, and more preferably 800 μm or less.

When the unit shape is linear, its width is preferably 10 mm or less, more preferably 8 mm or less, and still more preferably 5 mm or less, in order to allow interaction of humoral factors produced by adjacent cells. preferable. The lower limit can be appropriately set depending on the type of cells as long as the cells can be retained, and for example, 5 μm can be mentioned.

Furthermore, for reasons of interaction between cells, the shortest distance between adjacent unit shapes may be 1 μm or more, and may be 5 μm or more, or 10 μm or more. The upper limit can be appropriately set depending on the type of cells as long as the cells can be retained, and for example, 100 mm can be mentioned.

Examples of patterns formed by fine patterns include dots with circles arranged in rows, patterns with interspersed polygons, honeycomb shapes, lattice shapes (mesh shapes), scale shapes, geometric patterns, stripes, radial shapes, etc. Alternatively, a shape selected from the reverse pattern thereof is exemplified. Even if the pattern has continuity and symmetry, it may have any pattern without continuity or symmetry.

The average height (average thickness) of the fine pattern is preferably 10,000 nm or less, more preferably 1,000 nm or less, and even more preferably 500 nm or less. In addition, whether the unit shape is a non-cell adhesive substance or an exposed cell adhesive site, the ratio of the average height of the micropattern to the average circle equivalent diameter of the unit shape (average height/average circle The equivalent diameter) is preferably 0.5 or less, more preferably 0.1 or less, and even more preferably 0.01 or less. The lower limit can be appropriately set depending on the type of cells as long as the cells can be retained. For example, 0.00001 is given.

The proportion of the fine pattern of the cell non-adhesive substance on the surface of the substrate is preferably 5% or more, more preferably 10% or more, even more preferably 15% or more.

The surface of the micropattern only needs to be a cell non-adhesive site; for example, it may be formed by physically or chemically fixing a cell non-adhesive substance to the micropattern surface, and the micropattern itself may be a cell non-adhesive site. It may also be made of a non-adhesive material.

The method for manufacturing the base material 2 is not particularly limited, but for example, a fine pattern may be formed by printing a cell non-adhesive substance on a layer having a cell adhesive site.
Specifically, for example, a transfer mold having a desired fine pattern shape is prepared according to a known method, a cell non-adhesive substance is brought into contact with the patterned surface of the transfer mold, and a cell non-adhesive substance is applied to the surface of the layer having cell adhesive sites. It can be formed by printing (transferring) the patterned surface.

The method for forming the fine pattern is not particularly limited, and any known printing method can be used. Examples of printing methods include microcontact printing, spin coating, casting, roll coating, die coating, gravure coating, spray coating, bar coating, flexographic printing, dip coating, nanoimprinting, In addition to the inkjet method, a patterning method can be used in which a desired substance is injected into the gap by capillary force generated in the gap between the uneven structure formed on the surface of the base material.

For example, to form a fine pattern using the microcontact printing method, printing may be performed using a transfer mold having a fine pattern. There are no particular limitations on the method for preparing the transcription mold. Examples include methods for preparing known transfer molds such as elastomer stamps. Specifically, in addition to creating the desired shape as described above on an elastomer sheet such as a silicone material using a known processing machine and performing direct micromachining, we also create a mold using elastomer from a master substrate that has been microfabricated. There are several methods that can be used. When making a mold, first, a desired shape as described above is made on a substrate using a known processing machine and used as a master mold, an elastomer resin solution is introduced therein, and after curing, the master mold is The material obtained by peeling off is used as a replica mold. Here, the replica mold will have an inverted shape of the desired shape. Next, the replication mold is subjected to surface treatment if necessary, and then the resin solution is introduced again and after curing, the replication mold is peeled off to prepare a transfer mold having a desired shape. .

There are no particular limitations on the substrate used. For example, olefin resins (e.g. polyethylene, polypropylene), polyester resins (e.g. polyethylene terephthalate), polycarbonate resins, acrylic resins (e.g. polymethyl (meth)acrylate, styrene resins (e.g. polystyrene), polyether Examples include resins such as ketones, polyetheretherketones, and silicone resins; rubbers such as natural rubber and EPDM; glass; metal materials such as stainless steel; and ceramics. Since it has transparency etc., it can be used suitably.

The processing machine used for shaping the substrate is not particularly limited, and, for example, devices used for cutting with a drill, photolithography, inkjet printing, screen printing, and laser patterning methods are used. Specifically, for example, a fine excavation processing machine, a laser processing machine, etc. can be mentioned.

As the elastomer resin solution, any known elastomer resin solution can be used without particular limitation, and for example, from the viewpoint of the releasability of the transfer mold, a silicone material can be suitably used. The silicone material is preferably a curable silicone material such as silicone rubber, a curable silicone rubber oligomer or silicone monomer that becomes a silicone resin, or a curable silicone resin oligomer or monomer, and more preferably a curable polysiloxane. As the curable silicone material, what is usually used is what is called a liquid silicone, and from the viewpoint of excellent releasability and mechanical strength, a two-component mixture type used in combination with a curing agent is preferred. If a curable silicone material with low viscosity is used, it is possible to perform processability such as removing bubbles that are involved during production, and to make precise molding of the transferred pattern. Further, the curable polysiloxane may be a one-component curing type or a two-component curing type, and may be a thermosetting type or a room temperature curing type. As specific examples of curable silicone materials, those containing alkylsiloxanes, alkenylsiloxanes, alkylalkenylsiloxanes, and polyalkylhydrogensiloxanes are preferred, and among them, those containing a two-component mixed system of alkylalkenylsiloxanes and polyalkylhydrogensiloxanes with low From the viewpoint of viscosity and room temperature curing, it is preferable from the viewpoint of peelability and processability.

When the elastomer resin solution is introduced into the mold, defoaming treatment or the like may be performed according to a known method, if necessary.

Surface treatments for obtaining transfer replication molds include treatments to form a non-adhesive surface to facilitate mold release and improve transfer uniformity. For example, in addition to mold release treatment using fluorine-based solvents, etc., there is a treatment in which metals (Au, Pt, Ag, Ti, Al, Cr, Pd, etc.) or carbon are deposited on the surface and then coated. . Further, a treatment for reducing the remaining reactive functional groups may be performed.

The above-mentioned cell non-adhesive substance is attached to the thus obtained transfer-type fine pattern, and then the fine pattern of the cell non-adhesive substance is brought into contact (close contact) with the layer having the cell adhesive site and printed. With this method, a fine pattern can be formed in a thin film with high precision. Alternatively, the pattern can also be obtained by placing an elastomer pattern on the base material with the inverted mold taken in the above step, and injecting and forming a cell non-adhesive substance from the side using capillary action.

After the printing process, a known post-processing process such as drying the obtained base material may be included for the purpose of fixing the cell non-adhesive substance to the layer having the cell adhesive site.

The thickness of the base material is not particularly limited, but in the case of the above base material 1, it is preferably 10 to 5000 μm, more preferably 100 to 2000 μm, from the viewpoint of handleability.
Further, in the case of the base material 2, the thickness is preferably 1 to 1000 μm, more preferably 10 to 1000 μm, from the viewpoint of handleability and the like.

The area of the base material is also not particularly limited, but in the case of the above base material 1, it is, for example, 0.01 to 10,000 cm ² , preferably 0.03 to 5,000 cm ² .
Further, in the case of the base material 2, the thickness is, for example, 0.01 to 10,000 cm ² , preferably 0.03 to 5,000 cm ² .

<Cell>
The type of animal, organ, and tissue from which the cells are derived is not particularly limited, and can be appropriately selected depending on the purpose.

Specifically, for example, any organ or tissue (brain, liver, pancreas, spleen, heart, lung, intestine, cartilage, bone, fat, etc.) of a human or non-human animal (monkey, pig, dog, rat, mouse, etc.) Primary cells derived from tissue, kidney, nerve, skin, bone marrow, embryo, periosteum, synovium, muscle, dental pulp, placental tissue, umbilical cord tissue (cord blood), peripheral blood, etc.), established cell lines, or these Cells that have been genetically manipulated can be used.

More specifically, for example, ES cells, iPS cells, neural stem cells, mesenchymal stem cells, tissue stem cells (somatic stem cells), hematopoietic stem cells, cancer stem cells, and other undifferentiated stem cells or progenitor cells can be used. Stem cells may be cells derived from the above-mentioned organs or tissues, for example, cells derived from bone marrow, periosteum, synovium, adipose tissue, muscle, dental pulp, placental tissue, umbilical cord tissue (cord blood), peripheral blood, etc. It may be.
In addition, for example, cells derived from digestive system organs such as liver cells and pancreatic cells, cells derived from circulatory system organs such as kidney cells, nervous system cells, and cardiac muscle cells, adipocytes, and connective tissue such as skin dermis. differentiated fibroblasts, epithelial cells derived from epithelial tissues such as the skin epidermis, bone cells, cartilage cells, cells derived from ocular tissues such as the retina, vascular cells, blood cells, germline cells, etc. Cells can also be used. Furthermore, cancerous cells can also be used.
As such cells, one type of cell can be used alone, or two or more types of cells can be used in a mixture at any ratio.

The size of the retained cells can be appropriately selected depending on the structure of the substrate and is not particularly limited, but the diameter may be, for example, 10 to 1000 μm, preferably 10 to 800 μm. The diameter can be measured by a conventional method (eg, image analysis software, particle size distribution analyzer), and can be displayed as a fluid diameter or equivalent circle diameter, for example.

<Culture of cells>
The cells retained on the substrate are not particularly limited, and may be cells obtained by culturing separately, but from the viewpoint of reducing the influence on the cells as much as possible, cells cultured on the substrate may be used. It may be. That is, the substrate may be used not only for transportation but also for cell culture.
When holding cells cultured on a substrate, the cells used for culturing may, for example, be those that can form bonds with each other to form cell aggregates, and the cells may adhere to the cell adhesion site of the substrate. By culturing in such a manner, medium exchange is easy, and cell loss during medium exchange can be reduced, making it possible to easily culture cells.

The method for culturing cells on the substrate is not particularly limited, but, for example, cell culture may be carried out by placing a medium containing cells on the cell-adhesive site of the substrate.
In addition, the substrate is housed and fixed in various cell culture containers such as a culture dish (Petri dish), culture plate, culture flask, culture bag, etc., and a medium containing cells is added to the container to perform cell culture. You can. The method of fixing the substrate to the cell culture container is not particularly limited, and may be physical or chemical fixation.

The culture medium and conditions used when culturing cells can be appropriately set depending on the cells used. For example, the medium used may be appropriately selected depending on the cells. For example, any basic cell culture medium, a special medium developed for the target cells, a differentiation medium, a medium exclusively for primary culture, etc. can be used. Specifically, Eagle's small essential medium (EMEM), Dulbecco's modified Eagle's medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI1640, Ham's F-12, MCDB medium, Williams medium E, Hepatocyte thaw medium , and a mixed medium thereof. Any medium containing components necessary for cell proliferation and differentiation can be used. Furthermore, a medium containing serum, various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts, etc. may be used. Although the culture temperature is not particularly limited, it is usually carried out at about 25 to 40°C.

The cells obtained by culturing may be cells that have grown two-dimensionally, or may be cells that have formed three-dimensional or three-dimensional structures such as layers or spheroids. Preferably, it is spheroidal.

The size of the cells obtained by culture is not particularly limited, but the diameter is, for example, 10 to 1000 μm, preferably 10 to 800 μm.
In particular, when forming spheroids, the diameter of the spheroids is, for example, 10 to 1000 μm, preferably 10 to 800 μm. Here, the diameter can be measured by a conventional method (eg, image analysis software, particle size distribution analyzer), and can be displayed as, for example, a fluid diameter or an equivalent circle diameter. The spheroids have a circularity of, for example, 0.5 to 1.0, preferably 0.7 to 1.0.

Note that when culturing on the substrate, defoaming treatment may be performed in advance as necessary. The defoaming treatment is not particularly limited, and general treatments such as misting, pipetting, shaking, temperature changes such as heating and cooling, centrifugation, vacuum degassing, and ultrasonic treatment can be performed.
Defoaming treatment makes it difficult for air bubbles to form or remain in the substrate, especially when using a culture container with a substrate with a small diameter culture compartment (for example, the above substrate 1 or the above substrate 2). From the viewpoint of ease of culturing cells efficiently, etc., the method includes a step of feeding the liquid 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. It is preferable. An example of such a defoaming step is given below.

(Defoaming process)
The device or instrument used for liquid feeding is, for example, one having at least a liquid feeding port with 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, 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 culture compartment in the substrate (for example, a recess in the substrate 1, a unit shape having a compartment in the substrate 2), the diameter of the liquid feeding port and The diameter ratio (diameter of liquid feeding port/diameter of culture compartment opening) of the culture compartment opening (for example, the recess opening in substrate 1, the unit shape having compartments in substrate 2) is preferably 30 or less. , more preferably 20 or less, still more preferably 10 or less. The lower limit of the diameter ratio is not particularly limited, and may 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 liquid transfer, remove the liquid filled in the culture compartment after liquid transfer, and at that time, remove it to the extent that it does not completely disappear, and then replace it with the culture medium. By doing so, or by directly adding the culture solution after sending the solution, the culture compartment of the substrate can be filled with the culture solution. Moreover, before the liquid feeding, the liquid feeding liquid can be injected into the substrate in advance and then the liquid feeding can be performed, and when this pre-injection is performed, air bubbles may be confirmed in the culture compartment. . 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 it can flow out from the liquid feeding port and fill the inside of the culture compartment 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 culture compartment, 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 previously injected into the culture container in which the substrate is installed. The number of liquid feeding ports may be one or two or more per base material. Furthermore, the liquid may be fed to a plurality of culture compartments at once, or the liquid may be fed continuously or intermittently by moving the substrate itself or the liquid feeding port. Moreover, it may be automated or manual. Once the liquid is fed, the liquid present above the culture compartment may be sucked, and the liquid may be fed again above the same culture compartment or above a different culture compartment. 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, as long as the culture compartment can be filled with a liquid, the substrate can be subjected to known treatments required for cell culture (for example, changing the culture medium in a safety cabinet, standing still in an incubator), if necessary. good.

In this way, it is possible to manufacture a cell culture container filled with a culture solution in a state where bubbles are difficult to form or remain at least in the culture compartment. In addition, this defoaming process can remove bubbles that are formed when liquid is filled in a place where no liquid exists, or bubbles that are formed after being left for a while after filling. .

<Transport method>
The cell transport method is not particularly limited as long as it is a method of transporting cells while holding them on a base material having a cell-holding surface containing a polyimide resin. By using such a base material, it is possible to suppress cell collapse and aggregation due to vibrations during transportation, probably due to the appropriate adhesiveness between the cells and the base material.

Moreover, in the transport method of the present invention, it is preferable that after culturing cells on the above-mentioned substrate (particularly the cell-adhesive site of the substrate), the substrate holding the cells is transported as is.
For example, after using a cell culture container provided with the above-mentioned substrate and culturing cells on the substrate (particularly on the cell adhesion site of the substrate), the cell culture container may be transported as is. Note that after culturing, the cover of the cell culture container may be removed, and the outside and inside of the substrate or container, the cover, etc. may be disinfected with alcohol or the like, and then the cover may be attached to the cell culture container. Further, a cover film may be sandwiched between covers of the cell culture container.

The transport method is not particularly limited, and a commercially available cell transport system (or transport device) may be used. For example, cells can be transported by accommodating a substrate holding cells (or a cell culture container equipped with the substrate) in a cell transportation system (or transportation device).
Commercially available cell transport systems are not particularly limited, and include, for example, Cellporter (manufactured by Corefront).

Moreover, the transportation may be carried by a person or by various means of transportation.
The means of transportation is not particularly limited, and may be, for example, a railway (for example, a Shinkansen, a subway, etc.), an automobile, a ship, an airplane, or the like.

Further, the transportation may include one or more types of movement selected from accelerated movement and vibration. Such movement may be included temporarily during transportation.

Specific examples of the above movements include walking, running, climbing stairs, descending stairs, lifting, and placing. Transport may include one or more of these movements.

Note that during transportation, acceleration in the running direction (for example, 0.1 m/s ² or more) that is normally caused by walking may occur at least temporarily.

The transportation temperature (or the temperature inside the transportation device) is, for example, 25°C or higher, 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher. , 34°C or higher, 35°C or higher, 36°C or higher, or the like.
Further, the transportation temperature may be, for example, 40°C or lower, 39°C or lower, 38°C or lower, 37°C or lower, 36°C or lower, 35°C or lower, 34°C or lower, or the like.

Note that the transportation temperature may be in an appropriate range (for example, 25 to 37°C, 33 to 40°C, etc.) by appropriately combining these upper and lower limits.
In particular, the transport temperature may be 27 to 37°C, etc., from the viewpoint of little change from the cell culture temperature. In the transport method of the present invention, the substrate holding cells after culture can be transported as is in a transport device, so transport can be carried out at a transport temperature that does not change much from the cell culture temperature. Generally, cells become more dormant as the temperature decreases, but in the present invention, since transport can be carried out at a transport temperature that is less likely to change from the cell culture temperature, it is easy to prevent a decline in cell function due to transport.

The transportation time is not particularly limited, but may be, for example, 60 hours or less, 50 hours or less, 40 hours or less, 30 hours or less, 20 hours or less, 10 hours or less, 5 hours or less, 5 minutes or more, 30 hours or less, etc. The duration may be longer than minutes, longer than 1 hour, longer than 2 hours, longer than 3 hours, etc.

The transportation distance is not particularly limited, and may be, for example, 1 m or more, 5 m or more, 10 m or more, 100 m or more, 1 km or more, 10 km or more, 100 km or more, or 10,000 km or less, 1,000 km or less.

In the transport method of the present invention, the survival rate (viable cell rate) of cells after transport can be as high as, for example, 80% or more. In particular, when cells cultured on a substrate are transported as they are, the process from cell culture to transport can be performed continuously, so the survival rate of the cells after transport can be high.
Note that the method for calculating the viable cell rate is not particularly limited, and for example, the method described in Examples below may be used.

The transportation method of the present invention also includes a cell preservation step (or a cell preservation method).
The cell preservation step may be during the period during which the cells are transported, or may be during a period before, during or after the transport.

(Collection process)
The process for recovering the cells transported as described above is not particularly limited, and general processes such as pipetting can be performed.
The cell collection process is particularly effective when using a culture container with a culture compartment having a small diameter substrate (for example, the above-mentioned Substrate 1 or the above-mentioned Substrate 2). From the viewpoint of easily reducing damage to the collected cells, the liquid for feeding is fed to the substrate from a liquid feeding port with a diameter of 3 mm or less and/or at a flow rate of 100 cm/s or more. It is preferable to include a step of. An example of such a recovery process is given below.

The device or instrument used for liquid feeding during recovery is, for example, one that has at least a liquid feeding port with 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, 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 detaching and/or releasing cells from the culture compartment, the diameter of the liquid feeding port and the culture compartment opening (for example, the opening of the recess in the substrate 1, the The diameter ratio (diameter of liquid feeding port/diameter of culture compartment opening) of the unit shape having compartments in 2 is preferably 30 or less, more preferably 20 or less, still 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.

Further, it is preferable that the liquid feeding device or instrument for the recovery 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 in recovery is not particularly limited. Physiological saline, a buffer solution, or the like may be used, but the culture solution itself may also be used, or a culture solution that has been injected into a cell culture container in advance may be used.

The method of feeding the liquid during collection is not particularly limited, and may be such that it flows out from the liquid feeding port and detaches the cells in the culture compartment from within the culture compartment, or it may be such that the cells are released outside the culture compartment. 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 culture compartment, 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 may be one or two or more per base material. Furthermore, the liquid may be fed to a plurality of culture compartments at once, or the liquid may be fed continuously or intermittently by moving the substrate itself or the liquid feeding port. Moreover, it may be automated or manual.

Although there is no particular limitation on the liquid feeding flow rate during recovery, 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, 0.07 mL/s or more, and the upper limit is, for example, 20 mL/s.

Moreover, the liquid feeding flow rate in recovery is not particularly limited, and may 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. The upper limit includes a flow rate at which the cell non-adhesive substance on the substrate does not peel off or the substrate is not damaged, such as 2000 cm/s.

Furthermore, the liquid feeding during the recovery can be performed in a sterile environment. In the present invention, if cells can be detached and/or released from the culture compartment, cells will be present on the substrate in addition to the culture solution and the liquid feeding solution. By collecting the liquid present above, the cells themselves can be collected. There are no particular limitations on the means used to recover the liquid present on the substrate, as long as it does not apply strong force that would damage cells. For example, collection may be performed by suction or by pouring from a culture container. If necessary, additional fluid delivery may be performed to detach and/or release cells from the culture compartment. The collected cells can be subjected to known treatments (eg, passage, freezing, fixation, section preparation, flow cytometry evaluation, microscopic observation, genetic analysis, transplantation).

By the above-mentioned collection step, cells can be easily collected, and since the cells can be collected easily, it is possible to collect the cells with high efficiency. As described above, although cells can be transported while being held on the substrate according to the transport method of the present invention, it is unexpected that the cells can be easily detached from the substrate and collected in the above-mentioned collection step. It's outside.
In addition, according to the above-mentioned recovery step, since damage to cells is likely to be reduced, the viable cell rate in the recovered cell population is less likely to decrease from the viable cell rate in the cell population before recovery, for example, 80%. It can be much higher than that. Note that the method for calculating the viable cell rate is not particularly limited, and for example, the method described in Examples below may be used.

In addition, the transportation method of the present invention may include the above-mentioned defoaming step and/or the above-mentioned recovery step. That is, the transportation method of the present invention includes a defoaming step that includes a step of feeding the liquid feeding liquid 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, and/or The method may include a recovery step that includes a step of feeding the liquid feeding liquid to the substrate 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.

<Transport container>
The present invention also includes a container for transporting cells, which includes at least a base material having a cell-retaining surface containing a polyimide resin. It is preferable that the transport container holds cells on a cell holding surface.
The transportation container may be a container equipped with a base material having a cell-retaining surface containing polyimide resin (e.g., dish [petri dish (e.g., plastic petri dish, glass petri dish, etc.)], plate, flask, bag, etc.). good.
In the transport container, a base material having a cell-retaining surface containing polyimide resin may be installed or fixed within the container. Moreover, the base material (or the layer forming the base material) may be coated inside the container.
The transport container may also be provided with a lid or a seal (or film, cover film). A shipping container may include more than one lid or seal.
The transport container is preferably a container in which cells are cultured on a substrate contained in the container and then transported.

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.

Example 1
<Preparation of layer having cell adhesive site (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 166.6 g of N-methylpyrrolidone were charged and dissolved. A solution of 16.668 g (0.057 mol) of 1,4-bis(aminophenoxy)benzene and 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 15.0% by mass, 6FDA/TPEQ polyamic acid) was obtained. The weight average molecular weight of the polyamic acid was 120,000, and the viscosity was 6 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 83.0° and a falling angle of 24.0°.

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: Viscometer VISCOMETER TV-22 manufactured by As One
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 a viscometer calibration standard solution, measure using 0.3 g 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 the film, the adhesion angle of the droplet is measured (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 film, the base material is continuously tilted, and the angle at which the water falls is defined as the falling angle (measurement temperature: 25° C.).

<Preparation of layer having cell non-adhesive site>
After peeling the release tape on one side of the double-sided tape (thickness 25 μm), it was attached to a transparent PET film (thickness 250 μm), and then a CO ₂ laser was used 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 , 42,000 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 surface of the PET film with an MPC polymer solution (0.5% ethanol solution, hydrophobic MPC polymer) to a thickness of 0.05 μm. Spin conditions: 1000 rpm, 10 seconds) and dry in a dryer at 50°C for 2 hours to form a layer with cell non-adhesive areas [static water contact on the PET film coating layer (MPC polymer coating layer) side]. Angle 107.5°] was obtained.

<Preparation of substrate (cell culture sheet) and cell culture container>
Next, the layer having the cell-adhesive site prepared above is pasted on the side from which the other release tape of the double-sided tape of the layer having the cell-non-adhesive site has been removed, and the base material (cell culture sheet ) was prepared (sheet thickness: 315 μm). The obtained cell culture sheet was placed in a culture plate to complete a cell culture container.

<Defoaming treatment of culture containers>
The culture container obtained above was defoamed.
Specifically, in a safety cabinet, approximately 20 mL of PBS was first added by decant to a cell culture container. Next, fill a 20 mL all-plastic (manufactured by Nipro) with an 18G non-bevel needle (inner diameter 0.9 mm, manufactured by Terumo) attached with 20 mL of PBS, and place the tip of the needle in the PBS liquid in the container (recess opening). By extruding the syringe while moving it horizontally (approximately 2 mm above the upper 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/ ^cm2 of opening area). s, flow rate of approximately 393 cm/s), then suck up the PBS in the container with a syringe, refill the syringe with PBS, and then repeat the same liquid feeding to the remaining area to remove bubbles. went. 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, PBS was removed from the well so that it did not completely disappear, and then 21 mL of KBM ADSC-2 medium containing 1% antibiotics was added and the mixture was heated to 5% (v/v) at 37°C. v) It was left to stand overnight in a CO ₂ incubator.

Although residual air bubbles were found here and there in the recesses of the culture container before defoaming (non-defoaming), no air bubbles were observed in the recesses of the culture container that had been subjected to the defoaming process.

<Cell>
Human adipose derived stem cells (AdSC) were used as the cells. AdSC was purchased from a manufacturer (Lonza, PT-5006) and used.

<Cell expansion culture>
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 500×g for 5 minutes, the supernatant was removed and dispersed in 10 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 (manufactured by Sumitomo Bakelite) is 30 mL, and add the cell suspension to 1.0 x 10 ⁶ cells. / flask, and cultured (expanded culture) in a 5% (v/v) CO ₂ incubator at 37°C.

<Culture 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. 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 that had been subjected to the defoaming treatment, 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.
By using the culture container subjected to the above-described defoaming treatment, spheroids could be produced in almost all cavities. The container was photographed using a plate scanner (manufactured by Screen), and the obtained 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.

<Transportation>
After the culture, the cover of the culture container was removed and packed for transportation in a safety cabinet according to the following procedure.
(1) The outside, cover, inside, and upper end of the culture container were wiped with disinfectant alcohol.
(2) Two overlapping cover films were placed on top of the culture container, and the cover of the culture container was gently pushed in.
(3) It was confirmed that the medium did not leak from the culture container.
(4) The culture vessels subjected to the operations (1) to (3) above were placed in a Cellporter (manufactured by Corefront) container.
(5) After confirming that the CO ₂ gas concentration adjusting agent and data logger were in the container, the two heat storage agents were placed in the cellporter with the container sandwiched between them.

The culture container was placed in Cellporter as described above and maintained for 6 hours. During the 6-hour period, the subjects performed movements expected during transportation (walking, running, climbing stairs, lifting and placing the cell porter). After 6 hours, the medium slightly leaked between the outer wall of the culture container and the cover film, but the medium did not leak out of the culture container. In this way, in this example, a sufficient load was applied by performing the movements expected during transportation.

Moreover, FIG. 5 shows photographs of the culture container before and after transportation. In FIG. 5, black dots are spheroids, and only white circles are spheroids that have fallen off. As FIG. 5 shows, almost all spheroids remained attached within the cavity.
In addition, from the data logger record, the average temperature during transportation was 33°C.

Furthermore, when the spheroids were visually inspected after transportation, no collapse or aggregation was observed compared to before transportation.

<Collection of spheroids>
After the above transportation, the medium in the container was removed by suction, and 20 mL of PBS was added. Next, fill a 20 mL all-plastic (manufactured by Nipro) with an 18G non-bevel needle (inner diameter 0.9 mm, manufactured by Terumo) attached with 20 mL of PBS, and place the tip of the needle in the PBS liquid in the container (recess opening). By pushing out the syringe while horizontally moving it about 2 mm above the top end surface, the liquid is delivered to 1/3 of the entire concave part of the container in about 8 seconds (approx. 0.09 mL/s per ¹ cm2 of opening area, flow rate of about 393 cm) /s) to peel the spheroids from the substrate. The detached spheroids and PBS were sucked up with 20 mL of all plastic and collected into a 50 mL centrifuge tube.

Through the above recovery process, spheroids could be recovered without PBS leaking out of the container. In addition, spheroids could be collected without removing the container from the sterile environment and without using large equipment.

Furthermore, the survival rate of cells in the collected spheroids was measured as follows. The spheroid dispersion was centrifuged at 210×g for 5 minutes, suspended in 10 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 survival rate was calculated by counting the number of cells.

The viable cell rate in the collected spheroids was 90%.

The present invention can provide a method for transporting cells in which cell collapse is suppressed.

1 base material 11 recess
11a Inner surface of the recess 11b Bottom surface of the recess 12 Sheet surface 21 Substance exhibiting cell non-adhesion 22 Resin forming the cell adhesion site 3 Unit shape 4 Gap between adjacent unit shapes

Claims (10)

A cell transport method in which cells are held and transported on a base material having a cell-retaining surface containing polyimide resin,
the cell-retaining surface has a cell adhesive site containing a fluorine-containing polyimide resin,
The static water contact angle of the cell adhesion site is 70° or more,
The transportation time is more than 5 minutes,
A method for packaging and transporting a substrate having a cell-retaining surface containing polyimide resin. The method according to claim 1, wherein the transportation distance is 5 m or more. The method according to claim 1 or 2, wherein the transportation time is 30 minutes or more and the transportation distance is 10 m or more. The method according to any one of claims 1 to 3, wherein the cells to be retained on the substrate are cells cultured at a cell-adhesive site of the substrate. The method according to any one of claims 1 to 4, wherein the cell adhesion site of the substrate is divided by a wall, and the wall has a cell non-adhesion site. The base material 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 region, and the bottom surface of the recess has a cell adhesive region containing polyimide resin. The method according to any one of claims 1 to 5, which is a substrate having. 7. The method according to claim 1, wherein the cells are transported while forming spheroids. The method according to any one of claims 1 to 7, wherein the cells are transported while maintaining their culture environment. A method according to any of claims 1 to 8, wherein the method is transported at a temperature of 27 to 37°C. A defoaming process that includes a step of feeding the liquid feeding liquid from a liquid feeding port with a diameter of 3 mm or less and/or at a flow rate of 100 cm/s or more, and/or a defoaming process that includes feeding a liquid feeding liquid to the base material with a diameter of 3 mm or less. The method according to any one of claims 1 to 9, comprising a recovery step that includes a step of feeding the liquid feeding liquid from the following liquid feeding port and/or at a flow rate of 100 cm/s or more.