JP7317331B2 - Three-dimensional culture method, three-dimensional culture structure, and method for producing three-dimensional culture structure - Google Patents

Description

The present invention relates to a three-dimensional cell culture method (hereinafter referred to as "three-dimensional culture method"), a structure (including a container) used for three-dimensional culture, and a method for producing a three-dimensional culture structure.

In recent years, the three-dimensional cell culture method (hereinafter referred to as "three-dimensional culture method") has attracted attention as a technique indispensable for drug discovery and regenerative medicine (for example, Patent Documents 1 to 4, Non-Patent Documents 1 to 3).

The three-dimensional culture method is a method of culturing cells in vitro while allowing them to interact three-dimensionally, and it is possible to obtain spheroids that well reflect the properties of cells in vivo. Thus, the spheroids obtained by the three-dimensional culture method can express the properties and functions of the biological tissue from which the cultured cells are derived in a manner closer to that of the living body than the cells obtained by the two-dimensional culture method. . In the present specification, such a property of spheroids is referred to as "tissue reproducibility", and the tissue reproducibility is said to be high as the proteins produced by gene expression in cells function physiologically in a manner close to that of the living body. .

As a three-dimensional culture method, a culture method using a surface having a fine uneven structure is described in Patent Documents 1 to 3 and Non-Patent Document 1, for example. In the culture methods described in these documents, a cell suspension containing cells and a medium (here, it refers to a culture solution and a liquid medium) is poured into a container having a fine uneven structure on the bottom, and a portion of the cells is The part is cultured in the liquid while adhering to the bottom of the container. Hereinafter, the culture methods described in Patent Documents 1 to 3, Non-Patent Document 1, and the like are referred to as "microadhesion three-dimensional culture methods" in the present specification.

Also, a hanging drop method for culturing cells in droplets is described in Patent Document 4, Non-Patent Documents 2 and 3, for example.

JP 2005-168494 A WO2007/097120 WO2017/126589 WO2007/114351 Japanese Patent No. 4265729 JP 2009-166502 A WO2011/125486 WO2013/183576 WO2015/163018 JP 2010-226975 A

Yoshii Y, Furukawa T, Aoyama H, Adachi N, Zhang MR, Wakizaka H, Fujibayashi Y, Saga T,"Regorafenib as a potential adjuvantchemotherapy agent in disseminated small colon cancer: Drug selection outcome of anovel screening system using nanoimprinting 3-dimensional culture with HCT116-RFP cells", Int. J. Oncol., 2016 Apr;48(4):1477-84. Singla DK and Sobel BE, Biochem Biophys Res Commun. 2005 335(3):637-42 Foty, R., "A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids". JoVE., 51, 2720 (2011).

However, according to the studies of the present inventors, the conventional three-dimensional culture method described above still has room for improvement in terms of workability and mass productivity. In addition, development of a three-dimensional culture method capable of obtaining spheroids with further improved tissue reproducibility is desired.

In the micro-adhesion three-dimensional culture method using the fine uneven structure on the bottom surface, the fine uneven structure on the bottom surface acts as a scaffold for cells. If the interaction between the uneven structure of the bottom surface and the cells is stronger than the interaction between the cells, the cells cannot grow sufficiently in the thickness direction, and the growth in the in-plane direction becomes dominant. sufficient reproducibility of tissue structures. In addition, in the microadhesion three-dimensional culture method, the cells repeatedly contact and adhere to each other while randomly migrating in the in-plane direction, and form spheroids with cell division. There is also a problem that the reproducibility of the shape and size of the spheroids is low.

The hanging drop method has the advantages of easy control of the cell number and high reproducibility of the shape and size of the spheroids because the cells are cultured in droplets. However, cell types that are highly anchorage dependent may not maintain viability due to the lack of a cell scaffolding surface in the droplet. In addition, the hanging drop method has a problem of low workability because the surface on which the droplets are adhered faces downward (directs in the direction of gravity).

In addition, although the tissue reproducibility of spheroids obtained by any of the above three-dimensional culture methods is higher than that of spheroids obtained by two-dimensional culture method (flat culture method), further improvement is required. ing.

Therefore, an embodiment of the present invention provides a three-dimensional culture method capable of obtaining spheroids with superior workability or mass productivity and/or with high tissue reproducibility compared to conventional three-dimensional culture methods. With the goal. Another object of the present invention is to provide a three-dimensional culture structure suitable for use in such a three-dimensional culture method and/or a method for producing a three-dimensional culture structure.

According to embodiments of the present invention, solutions are provided in the following items.
[Item 1]
providing a cell suspension comprising cells and medium;
providing a solid surface formed from a porous alumina layer having a plurality of first recesses;
depositing droplets of the cell suspension onto the solid surface;
culturing the cells in the droplet with the direction of gravity acting on the droplet toward the solid surface.
[Item 2]
The surface of the porous alumina layer includes the plurality of first recesses, first ridgelines formed by intersections of side surfaces of the plurality of adjacent first recesses, and each of the plurality of first recesses. The three-dimensional culture method according to item 1, having a plurality of micro-recesses formed therein.
[Item 3]
The step of forming the porous alumina layer includes
Step (a) of forming a preliminary porous alumina layer by partially anodizing an aluminum film on a transparent substrate;
a step (b) of substantially completely removing the preliminary porous alumina layer after step (a) by contacting the preliminary porous alumina layer with an etchant;
After the step (b), a step (c) of anodizing the remaining aluminum layer of the aluminum film that has not been anodized,
The surface of the aluminum residual layer has a plurality of second recesses and second ridgelines formed by the intersection of side surfaces of the plurality of adjacent second recesses,
The plurality of first recesses reflects the plurality of second recesses, the first ridgeline reflects the second ridgeline,
3. The three-dimensional culture method according to item 2, wherein the step (c) includes a step of forming the plurality of micro recesses by anodizing the aluminum residual layer.
[Item 4]
4. The three-dimensional culture method according to item 3, wherein the anodizing step performed in the step (c) is performed using the same electrolytic solution as the anodizing step performed in the step (a).
[Item 5]
The step of forming the porous alumina layer includes
5. The three-dimensional culture method according to item 3 or 4, further comprising a step (d) of enlarging the plurality of micro-recesses by contacting the porous alumina layer with an etchant after the step (c). .
[Item 6]
The three-dimensional culture method according to item 5, wherein the etchant used in the step (d) is the same as the etchant used in the step (b).
[Item 7]
7. The three-dimensional culture method according to any one of items 3 to 6, wherein step (c) includes a step of substantially completely anodizing the remaining aluminum film.
[Item 8]
8. The three-dimensional culture method according to any one of items 1 to 7, wherein the transmittance of the porous alumina layer at a wavelength of 550 nm is 40% or more.
[Item 9]
9. The three-dimensional culture method according to any one of items 1 to 8, wherein the solid surface is subjected to release treatment with a fluorine-based release agent.
[Item 10]
The three-dimensional culture method according to item 9, wherein the fluorine-based release agent contains a fluorine compound having a perfluoroalkyl group.
[Item 11]
10. The three-dimensional culture method according to item 9, wherein the fluorine-based release agent contains a fluorine compound having a perfluoroalkyl group (-(CF ₂ ) _p - group, p is an integer of 0 or more and 2 or less).
[Item 12]
12. The three-dimensional culture method according to any one of items 1 to 11, wherein the solid surface has a water contact angle of 108° or more and 128° or less.
[Item 13]
13. The three-dimensional culture method according to any one of items 1 to 12, wherein the solid surface has a contact angle of 57° or more and 85° or less with respect to hexadecane.
[Item 14]
A three-dimensional culture structure having a solid surface for use in the three-dimensional culture method according to any one of items 1 to 13.
[Item 15]
A method of preparing a three-dimensional culture structure having the solid surface and manufacturing a three-dimensional culture structure having spheroids cultured using the three-dimensional culture method according to any one of items 1 to 13 on the solid surface. .
According to other embodiments of the present invention, solutions are also provided in the following items.
[Item 16]
preparing a cell suspension containing cells and a medium; preparing a solid surface having a plurality of protrusions with a height of 10 nm or more and 1 mm or less; and placing the cell suspension on the solid surface. A three-dimensional culture method, comprising the steps of attaching a droplet, and culturing the cell in the droplet with the direction of gravity acting on the droplet directed toward the solid surface.
[Item 17]
17. The three-dimensional culture method according to item 16, wherein the two-dimensional size of the plurality of protrusions is in the range of 10 nm or more and 500 nm or less when viewed from the normal direction of the solid surface.
[Item 18]
18. The three-dimensional culture method according to item 16 or 17, wherein the heights of the plurality of protrusions are 10 nm or more and 500 nm or less.
[Item 19]
19. The three-dimensional culture method according to any one of items 16 to 18, wherein the distance between adjacent protrusions is 10 nm or more and 1000 nm or less. A distance between adjacent protrusions may be 500 nm or less.
[Item 20]
20. The three-dimensional culture method according to any one of items 16 to 19, wherein the plurality of projections has a substantially conical tip portion.
[Item 21]
21. The three-dimensional culture method according to any one of items 16 to 20, wherein the solid surface has a contact angle of 17° or more with respect to the cell suspension. The contact angle of the solid surface with respect to the cell suspension should be 17° or more at least 10 seconds after droplet deposition.
[Item 22]
22. The three-dimensional culture method according to any one of items 16 to 21, wherein the solid surface has a contact angle of 90° or more with respect to the cell suspension. The contact angle of the solid surface with respect to the cell suspension should be 90° or more at least 10 seconds after droplet deposition.
[Item 23]
23. The three-dimensional culture method according to any one of items 16 to 22, wherein the sliding angle of the solid surface with respect to the cell suspension is 45° or more. The sliding angle can be evaluated by the value 20 seconds after the droplet is deposited.
[Item 24]
24. The three-dimensional culture method according to any one of items 16 to 23, wherein the solid surface is made of a synthetic polymer.
[Item 25]
25. The three-dimensional culture method according to any one of items 16 to 24, wherein the droplet has a volume of 10 μL or more and 50 μL or less.
The above ranges are preferable from the viewpoints of formation of droplets having a suitable shape, operability, and the like.
[Item 26]
26. The three-dimensional culture method according to any one of items 16 to 25, wherein the seeding density of the cells contained in the droplet is 10 ³ cells/mL or more and 10 ⁷ cells/mL or less.
[Item 27]
27. The three-dimensional culture method according to any one of items 16 to 26, wherein the droplet has a height of 1 mm or more.
[Item 28]
28. The three-dimensional culture method according to any one of items 16 to 27, further comprising applying the medium to the droplet while the cells are being cultured in the droplet.
[Item 29]
29. The three-dimensional culture method of item 28, further comprising blotting a portion of said medium from said droplet prior to applying said medium.
[Item 30]
A three-dimensional culture structure having a solid surface for use in the three-dimensional culture method according to any one of items 16-29.
A three-dimensional culture structure is provided as part of the container.
[Item 31]
A method of preparing a three-dimensional culture structure having the solid surface and manufacturing a three-dimensional culture structure having spheroids cultured using the three-dimensional culture method according to any one of items 16 to 29 on the solid surface. .
Spheroids cultured using the three-dimensional culture method of any of items 16-29 can be provided with a three-dimensional culture structure (eg, vessel).

According to embodiments of the present invention, a three-dimensional culture method is provided that is superior to conventional three-dimensional culture methods in terms of workability or mass productivity and/or can yield spheroids with high tissue reproducibility. In addition, according to another embodiment of the present invention, a three-dimensional culture structure suitable for use in such a three-dimensional culture method is provided. According to still another embodiment of the present invention, there is provided a three-dimensional culture structure (eg, container) having spheroids on its surface that have higher tissue reproducibility than conventional ones.

FIG. 2 is a diagram schematically showing culture conditions in a three-dimensional culture method according to an embodiment of the present invention; FIG. 3 is a schematic cross-sectional view of a synthetic polymer membrane 34A having a moth-eye structure on its surface, which is used in the three-dimensional culture method according to the embodiment of the present invention; FIG. 4 is a schematic cross-sectional view of a synthetic polymer membrane 34B having a moth-eye structure on its surface, which is used in a three-dimensional culture method according to an embodiment of the present invention; It is an optical microscope image (left) of the result of drop culture of the human liver cancer-derived cell line HepG2, and an optical microscope image (right) of the result of planar culture. A top view (left) and a side view (right) of electron microscope spheroids of HepG2 obtained by the drop culture method. FIG. 10 is a graph showing the results of determining the number of cell survival of hepatic cancer cell line HepG2 during drop culture. FIG. Fig. 10 is a graph showing the results of evaluating the CYP activity of HepG2 spheroids of liver cancer cells obtained by the drop culture method. It is an optical microscope image (left) of the result of drop culture of HEK293 human embryonic kidney epithelial cells, and an optical microscope image (right) of the result of planar culture. Fig. 2 shows an optical microscope image (left) of mouse preadipocyte 3T3-L1 drop culture and an optical microscope image (right) of planar culture. It is an optical microscope image (left) of the result of drop culture of mouse mesenchymal stem cells C3H10t1/2, and an optical microscope image (right) of the result of planar culture. It is an optical microscope image (left) of the result of drop culture of mouse myoblast C2C12, and an optical microscope image (right) of the result of planar culture. It is an optical microscope image of spheroids (level 1) obtained by the drop culture method. It is an optical microscope image of spheroids (level 2) obtained by the drop culture method. It is an optical microscope image of spheroids (level 3) obtained by the drop culture method. It is an optical microscope image of spheroids (level 4) obtained by the drop culture method. It is an optical microscope image of spheroids (level 5) obtained by the drop culture method. FIG. 4 is a schematic cross-sectional view for explaining the structure and formation method of the porous alumina layer used in the drop culture method. FIG. 4 is a schematic cross-sectional view for explaining the structure and formation method of the porous alumina layer used in the drop culture method. FIG. 4 is a schematic cross-sectional view for explaining the structure and formation method of the porous alumina layer used in the drop culture method. FIG. 4 is a schematic cross-sectional view for explaining the structure and formation method of the porous alumina layer used in the drop culture method. FIG. 4 is a schematic cross-sectional view for explaining the structure and formation method of the porous alumina layer used in the drop culture method. FIG. 3 is a schematic plan view for explaining the structure and formation method of the porous alumina layer used in the drop culture method. FIG. 3 is a schematic plan view for explaining the structure and formation method of the porous alumina layer used in the drop culture method. Sample sample no. 101 is an SEM image. Sample sample no. 102 is an SEM image. Sample sample no. 103 is an SEM image. Sample sample no. 104 is an SEM image. Sample sample no. 105 SEM image. It is an optical microscope image of spheroids obtained by the drop culture method (upper left: sample sample No. 101, upper right: sample sample No. 102, lower left: sample sample No. 103, lower right: sample sample No. 104).

A three-dimensional culture method, a three-dimensional culture structure, and a method for producing a three-dimensional culture structure according to embodiments of the present invention will be described below.

In the three-dimensional culture method according to the embodiment of the present invention, as schematically shown in FIG. In this method, cells 12C are cultured in droplets 16D with the direction of gravity acting on 16D directed toward solid surface 10S. In this three-dimensional culture method (hereinafter referred to as the "drop culture method"), cells 12C are cultured in droplets 16D, and thus, as in the hanging drop method, the number of cells can be easily controlled, and the shape and shape of spheroids can be easily controlled. The advantage is that the size is highly reproducible. Furthermore, since the culture is performed in a state where the direction of gravity acting on the droplet 16D is directed toward the solid surface 10S, the solid surface 10S acts as a scaffold. Maintain viability. In addition, since it is not necessary to direct the surface 10S onto which the droplet 16D is adhered downward (in the direction of gravity), workability is higher than that of the hanging drop method. The solid surface 10S has a plurality of protrusions 10Sp that can act as scaffolds.

The droplet 16D is in contact with the atmospheric gas (for example, air) except for the portion in contact with the solid surface 10S, forming a closed culture space. In FIG. 1, the bottom surface of the droplet 16D is in contact with the tip of the convex portion 10Sp, and the droplet 16D is illustrated as existing only above the tip of the convex portion 10Sp. may intrude between the adjacent protrusions 10Sp. The volume of the droplet 16D is, for example, 10 μL or more and 50 μL or less.

The solid surface 10S on which stable droplets 16D can be formed and cells can be efficiently cultured is a solid surface having a plurality of protrusions 10Sp with a height of 10 nm or more and 1 mm or less, as will be shown later in the experimental results. For example, Patent Document 1 (registered as Japanese Patent No. 4507845) describes that three-dimensional culture can be achieved by using a solid surface having a plurality of protrusions with a height of 10 nm or more and 1 mm or less. However, as described above, the microadhesion three-dimensional culture method that utilizes the fine uneven structure of the bottom surface has the problem that the three-dimensional tissue structure cannot be sufficiently reproduced, or the reproducibility of the spheroid shape and size is low. There is

Since the drop culture method cultures the cells in the droplet 16D, it is possible to overcome the above drawbacks of the microadhesion three-dimensional culture method. Cells 12C gather in a three-dimensionally closed droplet 16D to deposit on the bottom surface in contact with the solid surface 10S under the action of gravity. Therefore, there are a certain amount of cells that interact with the plurality of projections 10Sp of the solid surface 10S, and there are cells that interact only between cells thereon. As a result, it is thought that spheroids that grow moderately in the thickness direction and have a highly reproducible three-dimensional tissue structure can be obtained, unlike weak adhesion three-dimensional culture.

In the following, an experimental example using a solid surface 10S having a moth-eye structure will be shown to explain the three-dimensional culture method (drop culture method) according to the embodiment of the present invention. A solid surface with a moth-eye structure, which one of the applicants has developed as an antireflection film or a synthetic polymer film with bactericidal properties, is suitable for use in the drop culture method. For reference, the disclosures of Patent Documents 5 to 8 (antireflection film) and Patent Document 9 (synthetic polymer film having bactericidal properties) are incorporated herein by reference.

As described in Patent Documents 5 to 9, when an anodized porous alumina layer is used, a synthetic polymer film having a moth-eye structure on its surface (for example, formed by curing a photocurable resin) is highly mass-producible. A photocurable resin film formed by curing a thermosetting resin, a thermosetting resin film formed by curing a thermosetting resin, etc.) can be manufactured. Experimental examples shown below are examples using a photocurable resin film having a moth-eye structure on the surface formed by the above-described method, and have the characteristics described in items 2 to 9 above. However, as described in Patent Document 1, the size and height of the plurality of protrusions and the distance between adjacent protrusions (pitch when regularly arranged) are not limited to these. Conceivable. The material forming the moth-eye structure may be either an organic material or an inorganic material.

2A and 2B, the structures of synthetic polymer membranes 34A and 34B having a moth-eye structure on the surface used in the drop culture method will be described. Synthetic polymer membranes 34A and 34B are examples of three-dimensional culture structures according to embodiments of the invention.

2A and 2B show schematic cross-sectional views of synthetic polymer membranes 34A and 34B, respectively. The synthetic polymer films 34A and 34B illustrated here are both formed on the base films 42A and 42B, respectively, but are of course not limited to this. Synthetic polymer films 34A and 34B can be formed directly on the surface of any object.

The film 50A shown in FIG. 2A has a base film 42A and a synthetic polymer film 34A formed on the base film 42A. The synthetic polymer film 34A has a plurality of protrusions 34Ap on its surface, and the plurality of protrusions 34Ap form a moth-eye structure. When viewed from the normal direction of the synthetic polymer film 34A, the two-dimensional size _Dp of the convex portion 34Ap is within the range of 10 nm or more and 500 nm or less. Here, the "two-dimensional size" of the convex portion 34Ap refers to the area circle equivalent diameter of the convex portion 34Ap when viewed from the normal line of the surface. For example, when the convex portion 34Ap is conical, the two-dimensional size of the convex portion 34Ap corresponds to the diameter of the bottom surface of the cone. Further, a typical distance D _int between adjacent protrusions 34Ap is 10 nm or more and 1000 nm or less. As illustrated in FIG. 2A, when the convex portions 34Ap are arranged densely and there is no gap between the adjacent convex portions 34Ap (for example, the bottom surfaces of the cones partially overlap), the convex portions 34Ap The two-dimensional size _Dp is equal to the adjacent distance _Dint . A typical height _Dh of the convex portion 34Ap is 10 nm or more and 500 nm or less. There is no particular limitation on the thickness _ts of the synthetic polymer film 34A, as long as it is greater than the height _Dh of the projections 34Ap.

The synthetic polymer film 34A shown in FIG. 2A has a moth-eye structure similar to the antireflection films described in Patent Documents 5-8. In order to exhibit the antireflection function, it is preferable that the convex portions 34Ap are densely arranged without a flat portion on the surface. In addition, the convex portion 34Ap has a shape in which a cross-sectional area (a cross-section parallel to a plane perpendicular to the incident light beam, for example, a cross-section parallel to the surface of the base film 42A) increases from the air side toward the base film 42A side. A conical shape is preferred. Moreover, in order to suppress the interference of light, it is preferable to arrange the convex portions 34Ap without regularity, preferably at random. However, these features are not necessary when the synthetic polymer membrane 34A is used for drop culture. For example, the convex portions 34Ap do not need to be arranged densely, and may be arranged regularly. The upper and lower limits of D _p , D _int and D _h may also exceed the wavelength range of visible light, since there is no need to prevent reflection of visible light.

The film 50B shown in FIG. 2B has a base film 42B and a synthetic polymer film 34B formed on the base film 42B. The synthetic polymer film 34B has a plurality of protrusions 34Bp on its surface, and the plurality of protrusions 34Bp form a moth-eye structure. In the film 50B, the structure of the projections 34Bp of the synthetic polymer film 34B is different from the structure of the projections 34Ap of the synthetic polymer film 34A of the film 50A. The description of features common to the film 50A may be omitted.

When viewed from the normal direction of the synthetic polymer film 34B, the two-dimensional size _Dp of the convex portion 34Bp is in the range of 10 nm or more and 500 nm or less. A typical adjacent distance D _int of the protrusions 34Bp is 10 nm or more and 1000 nm or less, and D _p <D _int . That is, in the synthetic polymer film 34B, flat portions exist between adjacent convex portions 34Bp. The convex portion 34Bp has a cylindrical shape with a conical portion on the air side, and a typical height _Dh of the convex portion 34Bp is 10 nm or more and 500 nm or less. Moreover, the convex portions 34Bp may be arranged regularly or may be arranged irregularly. When the convex portions 34Bp are arranged regularly, _Dint also represents the period of the arrangement. This is, of course, the same for the synthetic polymer membrane 34A.

In this specification, the "moth-eye structure" is composed of protrusions having a shape in which the cross-sectional area (the cross section parallel to the film surface) increases, such as the protrusions 34Ap of the synthetic polymer film 34A shown in FIG. 2A. It not only has a nano surface structure with an excellent antireflection function, but also has a portion with a constant cross-sectional area (cross section parallel to the film surface) like the convex portion 34Bp of the synthetic polymer film 34B shown in FIG. 2B It contains a nano-surface structure composed of protrusions. The tip of the protrusion does not necessarily have to be conical.

Although the plurality of protrusions on the solid surface illustrated in the examples has a substantially conical tip, the shape of the plurality of protrusions is not limited to this. However, when forming a plurality of protrusions using a mold, from the viewpoint of releasability, it is preferable that the tips of the protrusions are thinner (the closer to the bottom of the recess of the mold, the thinner). The tip need not be sharp. On the other hand, if the height of the protrusions (the depth of the recesses of the mold) exceeds 500 nm, there are disadvantages such as a decrease in releasability or a long time required to manufacture the mold.

The surfaces of the synthetic polymer membranes 34A and 34B may be treated as necessary. For example, a water- and oil-repellent agent or a surface treatment agent may be applied in order to adjust the surface tension (contact angle of the drop). A thin polymer film is formed on the surfaces of the synthetic polymer films 34A and 34B depending on the type of water/oil repellent and surface treatment agent. Also, the surfaces of the synthetic polymer films 34A and 34B may be modified using plasma or the like. For example, plasma treatment can impart lipophilicity to the surfaces of synthetic polymer membranes 34A and 34B.

A mold for forming a moth-eye structure on the surface as exemplified in FIGS. 2A and 2B (hereinafter referred to as "moth-eye mold") has an inverted moth-eye structure. If the anodized porous alumina layer having the inverted moth-eye structure is used as it is as a mold, the moth-eye structure can be manufactured at low cost. In particular, when a cylindrical moth-eye mold is used, the moth-eye structure can be efficiently manufactured by a roll-to-roll method. Such a moth-eye mold can be produced by the methods described in Patent Documents 5-8.

The method for manufacturing the moth-eye mold is not limited to the above method. For example, various lithography methods such as interference exposure lithography and electron beam lithography, or known nanostructure forming methods such as a method of forming a structure by irradiating a glassy carbon substrate with an oxygen ion beam can be used.

The effect of the interaction between the solid surface and the cell (or the action of the solid surface as a scaffold) on spheroid formation varies depending on the cell type, so there are many things that remain unknown until future research, but at least this is the case. From the experimental results up to this point, solid surfaces having the characteristics described in items 2 to 9 above are suitable for use in the drop culture method.

In experimental examples shown later, synthetic polymer membranes described in Japanese Patent Application No. 2018-041073 (filed on March 7, 2018) and US Application No. 16/293,903 based thereon were used. The entire disclosure of the above patent application is incorporated herein by reference. It should be noted that the synthetic polymer membranes described in the above patent application have the characteristic that the pH of the droplets adhering to the surface does not change. Specifically, 5 minutes after 200 μL of water is dropped on the surface of the synthetic polymer membrane, the pH of the aqueous solution may be 6.5 or more and 7.5 or less. In a synthetic polymer film produced using a photocurable resin, an acid generated due to a polymerization initiator may be eluted into water adhering to the surface. In order to prevent this, a polymerization initiator such as ethanone, 1-[9-ethyl-6-(2-methylbenzoyl)-9H-carbazol-3-yl]-,1-(O-acetyloxime ), 2-hydroxy-1-{4-[4-(2-hydroxy-2-methyl-propionyl)-benzyl]phenyl}-2-methyl-propan-1-one, and 1-[4-(2- One or more polymerization initiators selected from the group consisting of hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one may be used. Specifically, IRGACURE OXE02 (BASF), Omnirad 127 (IGM Resins), and Omnirad 2959 (IGM Resins) can be exemplified.

Excessive changes in pH of droplets adhered to a solid surface may result in a decrease in cell growth rate, inconsistent spheroid morphology, or reduced tissue reproducibility of spheroids. From this point of view as well, the synthetic polymer membrane described in the above patent application is preferably used.

FIG. 3A shows images of the results of drop culture of the human liver cancer-derived cell line HepG2 (left) and the results of planar culture (right) observed using an inverted phase-contrast microscope. In addition, FIG. 3B shows an electron microscope image of HepG2 spheroids obtained by the drop culture method. The left of FIG. 3B is a top view and the right of FIG. 3B is a side view.

Drop culture was performed by the following method.

First, human liver cancer-derived cell line HepG2 cells were cultured in a medium (cell culture solution) obtained by adding 10% final concentration of fetal bovine serum (FBS) to Dulbecco's modified Eagle's medium (D-MEM), which is a general culture condition. Atmospheric conditions of temperature 37° C., carbon dioxide concentration 5%, and relative humidity 95%, maintenance culture was performed in a petri dish for adherent cell culture (for example, MS-11600 manufactured by Sumitomo Bakelite Co., Ltd.).

Next, the HepG2 cells that had been subjected to this maintenance culture are detached from the culture dish using a general cell detachment solution, trypsin solution, and the cell density in the state in which the cells are suspended is measured by a fully automatic cell counter (cell counter). ), and a cell suspension was prepared in the above medium so as to have 1×10 ⁵ cells/mL. 25 μL of this cell suspension was weighed and attached to the surface of the photocurable resin film having a moth-eye structure on the surface so as to form droplets. The optimal number of droplets was 6 to 9 on the nano-projection film pasted on a 35 mmφ dish. This droplet (25 μL) was cultured for 3 days under the same atmospheric conditions as above (temperature 37° C., carbon dioxide concentration 5%, relative humidity 95%). The droplets maintained their shape even in this atmospheric condition and even when half or all of the medium was replaced to adjust the osmotic pressure in the medium.

As shown on the left side of FIG. 3A, when drop culture was performed on a surface having a moth-eye structure, three-dimensional spheroids having a generally circular outer circumference were formed. The formation of three-dimensional spheroids can also be confirmed from the electron microscope image in FIG. 3B.

On the other hand, the right side of FIG. 3A shows the results of general planar culture. No spheroid formation was observed on the right side of FIG. 3A. In addition, general planar culture was performed as follows here.

Prepared in a culture petri dish (for example, MS-3096 or MS-11350 manufactured by Sumitomo Bakelite Co., Ltd., selected according to the volume for the test) with a general cell adhesion surface coat (hydrophilic coat, etc.) An appropriate amount (100 μL for a 96-well plate, 2 mL for a 35-mm dish) of the obtained cell suspension was seeded and cultured so that the cells could grow in a plane.

FIG. 3C shows the results of determining the cell survival number of the liver cancer cell line HepG2 during drop culture. Here, quantification was performed by measuring adenosine triphosphate (ATP), which is known to have the same amount of energy in living cells of the same cell line. 3D in FIG. 3C shows the results of the drop culture method, and 2D shows the results of the general flat culture method.

As can be seen from FIG. 3C, the cell viability by the drop culture method is almost the same as the cell viability by the flat culture method. In the conventional three-dimensional culture method (for example, Patent Document 1), the number of viable cells is lower than that in the planar culture method. said to be alive. The number of viable cells depends on the cell density, but at a typical seeding density of 1 × 10 ⁵ cells/mL, it was found that the drop culture method yields almost the same cell viability as the flat culture method. rice field.

FIG. 3D shows the results of evaluating tissue reproducibility (or “gene expression”) of hepatic cancer cell HepG2 spheroids obtained by the drop culture method. 3D in FIG. 3D shows the results of the drop culture method, and 2D shows the results of the general flat culture method.

In HepG2 cells, hepatocyte functions are hardly maintained in planar culture, but by performing three-dimensional culture, cell coordination according to polarity becomes possible, and drug-metabolizing enzymes, which are one of the characteristic hepatic functions, become possible. It is known to restore cytochrome P450 activity (hereinafter sometimes abbreviated as “CYP activity”), and is used as one of the indices for evaluating tissue reproducibility of cultured spheroids. Therefore, tissue reproducibility of hepatocyte spheroids obtained by the drop culture method was evaluated by measuring P450 activity as follows.

Using the spheroids obtained by the drop culture method, enzyme activity in cells was measured using P450-Glo ^™ Luciferin-IPA kit (manufactured by Promega) according to the instructions. At this time, for comparison, HepG2 cells were plate cultured so as to have the same number of cells as the droplets, and the P450 activity was measured by the same method. Since it was expected that the cell growth rate would be different between the plate culture and the drop culture, it is necessary to calculate the correction value of the enzyme activity per cell. Therefore, in order to measure the number of viable cells when drop culture or plate culture was performed under the same conditions as when measuring P450 enzyme activity, ATP was quantified using the Cell Titer Glo ^R kit (manufactured by Promega). , RLU values were measured according to the instructions. The P450 enzymatic activity in flat culture or drop culture was divided by the ATP value, and the relative P450 enzymatic activity value of HepG2 cells in drop culture was obtained when the enzyme activity value per cell in flat culture was set to 1. The result is shown in FIG. 3D.

As can be seen from FIG. 3D, in the HepG2 spheroids formed by the drop culture method, the CYP activity increased about 10-fold after 3 days of culture, suggesting that the formed spheroids have high tissue reproducibility. be done.

From the above, it was found that spheroids with high tissue reproducibility can be formed by forming droplets on a solid surface and culturing them.

FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D show the results of drop culture of various cells (left) together with the results of planar culture (right). FIG. 4A shows an optical microscope image (left) of the result of drop culture of HEK293 human embryonic kidney epithelial cells and an optical microscope image (right) of the result of planar culture. FIG. 4B shows mouse preadipocyte 3T3-L1. An optical microscope image (left) of the results of drop culture and an optical microscope image (right) of the results of planar culture are shown. FIG. ) and an optical microscope image (right) of the results of planar culture, and FIG. ).

As can be seen from the results of FIGS. 4A, 4B, 4C, and 4D, spheroid formation was confirmed by the drop culture method, whereas no spheroids were formed by the flat culture method. As understood from this, the drop culture method is suitable for culturing a wide range of cell types.

Next, the results of examination of solid surfaces suitable for use in the drop culture method will be described.

[Synthetic polymer membrane]
(Experimental example 1)
A sample film having a structure similar to that of the film 50A shown in FIG. 2A was produced using ultraviolet curable resins having different compositions. Table 1 shows the raw materials used for the ultraviolet curable resin forming the synthetic polymer film 34A of each sample film, and Table 2 shows the composition of the ultraviolet curable resins A, B and C. Resins A, B and C are each mixed with a fluorine-based water- and oil-repellent agent (water-repellent additive).

The mold for forming the moth-eye structure on the surface used a porous alumina layer produced by the method described in Patent Documents 5 to 8 and Japanese Patent Application No. 2018-041073. As a flat "mold", non-alkali glass (EAGLE XG manufactured by CORNING) with a thickness of 0.7 mm was used.

Further, when forming the synthetic polymer film 34A, each mold was subjected to the mold release treatment shown in Table 3. Three types of treatment were performed by changing the concentration of fluorine-based release agent UD509 (OPTOOL UD509, modified perfluoropolyether manufactured by Daikin Industries, Ltd.).

The contact angle was measured as a parameter that characterizes the surface of molds and synthetic polymer membranes (solid surfaces in the drop culture method). Table 3 shows the contact angles on the surfaces of the molds. The contact angle of the solid surface with respect to the cell suspension affects the contact area between the solid surface and the cells (also referred to as the "bottom area of the droplet") and the shape of the droplet. Depending on the cell type, the shape of the spheroid obtained changes depending on the contact angle. It is preferable to adjust the contact angle according to the cell type.

The contact angle (static contact angle) is measured by a general θ/2 method (half-angle Method: (θ/2 = arctan (h/r), θ: contact angle, r: droplet radius, h: droplet height)). For the contact angle measurement using pure water, droplets of 10 μL to 70 μL were used considering the volume of droplets used for 1 μL and the drop culture method. For contact angle measurement using a medium, droplets of 10 μL to 70 μL were used in consideration of the droplet volume used in the drop culture method. The contact angle changes depending on time. Therefore, the contact angle was measured 1 second and 10 seconds after the droplet was applied. The contact angle for characterizing a solid surface refers to the static contact angle 10 seconds after the droplet adheres to the solid surface. In addition, "no drop" means that the contact angle is 140° or more. In addition, for the measurement of the sliding angle, droplets of 10 μL to 70 μL were used as in the measurement of the contact angle. The sliding angle is the inclination angle at which the droplet starts to slide downward when the surface on which the droplet adheres is tilted from the horizontal direction.

D-MEM (Low Glucose 1.0 g/L Glucose)/10% FBS was used as the medium. The influence of the type and concentration of the medium on the contact angle and sliding angle was within the range of variation. The effect of adding cells to the medium on the contact angle was also within variability.

Table 4 shows the mold (type of release treatment) and resin composition used to prepare the synthetic polymer membranes (Comparative Examples 1 to 12, Examples 1 to 12) used in the experiment, and the surface of each synthetic polymer membrane. The results of measuring the contact angle with water are shown. The contact angle was measured using 1 μL of pure water. The results of measurement of the contact angle 1 second and 10 seconds after the droplet was attached to the surface and the difference (Δ) obtained by subtracting the contact angle after 1 second from the contact angle after 10 seconds are shown.

As can be seen from Table 4, although there are some variations, the higher the water repellency, the higher the synthetic polymer film produced using the mold treated with the high-concentration release agent. When a flat surface and a surface having a moth-eye structure (hereinafter referred to as a moth-eye surface) are compared, the moth-eye surface has a larger contact angle and higher water repellency (lotus effect). On the moth-eye surfaces of Examples 1, 2, and 3, even when a droplet formed on the tip of the needle is brought into contact with the moth-eye surface when measuring the contact angle, the droplet does not adhere to the moth-eye surface and remains on the tip of the needle. Therefore, the contact angle could not be measured. The phenomenon occurred that the droplet thus failed to adhere to the surface of the object when the contact angle exceeded approximately 140°.

Moreover, looking at the change over time (Δ) of the contact angle, except for Examples 10 and 11, the change over time of the contact angle is small and stable. The reason why the contact angle of Examples 10 and 11 changed significantly over time is considered to be that the concentration of the release agent was low and the water and oil repellent was not evenly and sufficiently attracted to the moth-eye surface. This is because the water and oil repellent contained in the curable resin is attracted to the moth-eye surface by the release treatment of the mold.

Tables 5, 7, 9, and 11 show the results of measuring the contact angle to water and the contact angle to the culture medium while varying the amount of droplet (volume of droplet). Table 5 (Comparative Examples 1-1 to 12-1, Examples 1-1 to 12-1) shows the results using 10 μL droplets, and Table 7 (Comparative Examples 1-2 to 12-2, Examples 1-2 to 12-2) show the results using 30 μL droplets, and Table 9 (Comparative Examples 1-3 to 12-3, Examples 1-3 to 12-3) show the results using 50 μL droplets. Table 11 (Comparative Examples 1-4 to 12-4, Examples 1-4 to 12-4) shows the results using 70 μL droplets.

The contact angle of the moth-eye surface is larger than that of the flat surface for both water and the medium, and the water repellency is higher. As the droplet volume increases, the droplet shape tends to flatten under the influence of gravity, and the contact angle tends to decrease for water and medium. In addition, although there are variations, it is recognized that synthetic polymer films produced using a mold treated with a release agent having a higher concentration tend to have higher water repellency.

The sliding angle also tends to decrease under the influence of gravity as the droplet volume increases for water and medium. The sliding angle is larger on the moth-eye surface than on the flat surface, and is thought to be due to the action of fine convex portions on the moth-eye surface. That is, it can be seen that the moth-eye surface can maintain a high sliding angle while having high water repellency.

Table 6 (Comparative Examples 1-1 to 12-1, Examples 1-1 to 12-1) and Table 8 (Comparative Example 1- 2 to 12-2, Examples 1-2 to 12-2), Table 10 (Comparative Examples 1-3 to 12-3, Examples 1-3 to 12-3), Table 12 (Comparative Examples 1-4 to 12-4, Examples 1-4 to 12-4).

Evaluation of spheroid formation was based on observation of morphology with an optical microscope. The level of spheroid formation was evaluated on a scale of 5, and it was determined that the higher the number of the level, the better the state of spheroid formation (high density accumulation). Examples of morphological observation results by an optical microscope are shown in FIG. 5A (level 1), FIG. 5B (level 2), FIG. 5C (level 3), FIG. 5D (level 4), and FIG. 5E (level 5). In Tables 6, 8, 10 and 12, ◯ (good) indicates that spheroids of level 3 or higher were formed, and Δ (acceptable) indicates that spheroids of level 2 or 1 were obtained, and no spheroids were observed. x (impossible). FIG. 5A is Example 10-4, the left of FIG. 5B is Example 10-3, the center is Example 11-4, the right is Example 10-1, the left of FIG. 5C is Example 9-2, and the right is Example Example 8-4, FIG. 5D is Example 6-2, FIG. , indicates the change in contact angle (▼ indicates minus).

The workability of medium exchange was evaluated by the contact angle. A contact angle of 110° or more was evaluated as ⊚ (excellent), a contact angle of 90° or more and less than 110° was evaluated as ◯ (good), and a contact angle of less than 90° was evaluated as Δ (acceptable). However, if the height of the droplet is less than 1 mm, the workability of the medium exchange is lowered, so it was marked as x (impossible). Since the drop culture method has high viability, the culture period may be long (exceeding several days) depending on the cell type. As a result, the medium in the droplet is reduced by evaporation. Also, waste products in the droplets are increased. Therefore, it is preferable to perform a step of applying the medium to the droplet, and further a step of sucking part of the medium from the droplet before applying the medium. In order to efficiently perform such an operation of exchanging the medium using a dispenser, the droplet height is preferably 1 mm or more. The contact angle obtained by the θ/2 method is obtained by assuming that the shape of the droplet is part of a circle (θ/2 = arctan (h/r), θ: contact angle, r: radius of the droplet, h: droplet height). From this relationship, for example, when the droplet volume is 70 μL and the contact angle is 17°, the height h is 1 mm (20° for 50 μL, 26° for 30 μL, and 44° for 10 μL). °, each with a height h of 1 mm). Therefore, only Example 10-4 with medium contact angles of 14.5° and less than 17° after 10 seconds was evaluated as x.

The handleability was evaluated by the slide angle, which indicates how easily the liquid droplet can be stably held on the solid surface during operation. A droplet on a solid surface may move (slide or roll) when the solid surface is tilted or vibrated. In order to prevent this, it is necessary to maintain a state in which the solid surface is not tilted or vibrated during culture, which reduces workability. For example, by setting the sliding angle of the solid surface to the culture medium to 45° or more, the liquid droplet can be held relatively stably on the solid surface. The handleability was evaluated as ◎ (excellent) when the sliding angle was 90 ° or more, ○ (good) when 45 ° or more and less than 90 °, △ (acceptable) when 10 ° or more and less than 45 °, and × (improper) when the sliding angle was less than 10 °. .

Looking at the evaluation results of spheroid formation in Tables 6, 8, 10, and 12, no spheroid formation was observed in any of the comparative examples using the flat surface, whereas all the samples using the moth-eye surface showed no formation of spheroids. It can be seen that the formation of spheroids was observed in Examples. Moreover, in the examples, as can be seen from FIGS. 5A to 5E, there is a tendency that the larger the contact angle, the better the spheroids obtained. This is probably because the larger the contact angle, the closer the shape of the droplet is to a sphere, and the more densely the cells accumulate on the bottom surface of the droplet. The contact angle is preferably at least 17° or more, more preferably 90° or more. As for the contact angle, the value 10 seconds after droplet deposition satisfies the above conditions.

Further, as can be seen by comparing Example 11-4 (center of FIG. 5B) and Example 10-1 (right of FIG. 5B) with Example 8-4 (right of FIG. 5C), the contact angle difference Δ is small. A tendency to obtain spheroids in good condition is recognized. This is because the smaller the contact angle change in 10 seconds after droplet deposition, the easier it is for the droplet shape to be maintained during the culture period (the less likely it is to be flattened). , is considered to have been obtained more.

From the viewpoint of droplet formation and operability, etc., the droplet volume is preferably 10 μL or more and 50 μL or less (see Tables 6, 8 and 10, especially Examples 1 to 6).

The seeding density of the cells contained in the droplet is, for example, 10 ³ cells/mL or more and 10 ⁷ cells/mL or less. One advantage of the drop culture method is that the number of cells contained in the droplet can be precisely controlled. The number of cells is typically within the above range, but can be adjusted as appropriate according to the cell type, droplet volume, and the like.

As described above, from the viewpoint of handleability, the sliding angle of the droplet is preferably 45° or more, more preferably 90° or more. The sliding angle can be evaluated by the value 20 seconds after the droplet is deposited.

[Anodized porous alumina layer]
In Experimental Example 1 described above, a photocurable resin film having a moth-eye structure on the surface was used, but below, as an example of a different solid surface, an example using an anodized porous alumina layer having a plurality of recesses on the surface is shown.

The structure and formation method of the porous alumina layer used in the drop culture method will be described with reference to FIGS. 6A to 6E, 7A and 7B. 6A to 6E, 7A and 7B illustrate an example in which the concave portions of the porous alumina layer form a highly regular (periodic) arrangement for the sake of simplicity. However, it is not limited to such an example. As can be seen from SEM images (FIGS. 8A to 8E) of specimen samples shown later, here, the concave portions 64p1 and the micro concave portions 64p2 of the porous alumina layer 64 are arranged irregularly (no periodicity). ). The recesses (pores) of the porous alumina layer form a highly regular (periodic) arrangement under certain conditions, and depending on the conditions, a somewhat disordered arrangement or an irregular (periodic) arrangement. are known to form sequences that do not have When the porous alumina layer is used in the drop culture method, no significant difference (difference in cell culture results) due to whether or not the arrangement of the recesses has regularity is observed.

First, as shown in FIG. 6A, a transparent substrate (transparent base material) 62 and an aluminum film 68 formed on the transparent substrate 62 are prepared.

The transparent substrate 62 is, for example, a glass substrate, a plastic substrate, or the like, and may have a visible light transmittance of 90% or more. The thickness of the transparent substrate 62 is, for example, 0.03 mm or more and 1 mm or less.

The aluminum film 68 is, for example, a film made of aluminum with a purity of 99.99 mass % or more (hereinafter sometimes referred to as a "high-purity aluminum film"), as described in Patent Document 7. The aluminum film 68 is formed using, for example, a vacuum deposition method or a sputtering method. The thickness of the aluminum film 68 is, for example, 100 nm or more and 1 μm or less.

Further, as the aluminum film 68, instead of the high-purity aluminum film, an aluminum alloy film described in Patent Document 8 may be used. The aluminum alloy film described in Patent Document 8 contains aluminum, a metal element other than aluminum, and nitrogen. In this specification, "aluminum film" includes not only the high-purity aluminum film but also the aluminum alloy film described in Patent Document 8.

By using the aluminum alloy film, a mirror surface with a reflectance of 80% or more can be obtained. The average grain size of the crystal grains forming the aluminum alloy film when viewed from the normal direction of the aluminum alloy film is, for example, 100 nm or less, and the maximum surface roughness Rmax of the aluminum alloy film is 60 nm or less. The nitrogen content in the aluminum alloy film is, for example, 0.5 mass % or more and 5.7 mass % or less. The absolute value of the difference between the standard electrode potential of metal elements other than aluminum contained in the aluminum alloy film and the standard electrode potential of aluminum is 0.64 V or less, and the content of the metal elements in the aluminum alloy film is 1.0 mass. % or more and 1.9 mass% or less. A metal element is, for example, Ti or Nd. However, the metal element is not limited to this, other metal elements (for example, Mn, Mg, Zr, V and Pb). Furthermore, the metal element may be Mo, Nb or Hf. The aluminum alloy film may contain two or more of these metal elements. The aluminum alloy film is formed by DC magnetron sputtering, for example. The thickness of the aluminum alloy film is preferably in the range of 100 nm or more and 1 μm or less.

Next, as shown in FIG. 6B, a preliminary porous alumina layer 69 is formed by partially anodizing the aluminum film 68 . Here, only a portion of the aluminum film 68 including the surface 68s of the aluminum film 68 is anodized. That is, the preliminary porous alumina layer 69 is formed on the remaining aluminum layer 68r of the aluminum film 68 that has not been anodized. The preliminary porous alumina layer 69 has a porous layer 69a having recesses (pores) 69p and a barrier layer 69b (bottom of the recesses (pores) 69p). The thickness of the porous layer 69a corresponds to the depth Ddp of the recess 69p.

The preliminary porous alumina layer 69 is then substantially completely removed by contacting the preliminary porous alumina layer 69 with an etchant, as shown in FIG. 6C. The surface of the remaining aluminum layer 68 r reflects the shape of the bottom of the barrier layer 69 b of the preliminary porous alumina layer 69 . The surface of the remaining aluminum layer 68r has a plurality of recesses 68p and ridgelines 68d formed by the intersection of the side surfaces of the adjacent recesses 68p.

The shapes of the preliminary porous alumina layer 69 and the remaining aluminum layer 68r will be described with reference to the plan view of FIG. 7A. When viewed from the normal direction of the surface, the centers 63o of the recesses 69p are arranged, for example, in an equilateral triangular lattice. The recess 69p is shown as a circle around the center 63o in plan view. Here, the recess 69p is cylindrical and has a bottom with a U-shaped cross section. Adjacent circles of circles around the center 63o indicating the recesses 68p of the aluminum remaining layer 68r are overlapped with each other to form a ridge line 68d between the adjacent recesses 68p. The ridgeline 68d is a side of a regular hexagon inscribed in a circle when the recess 68p is viewed from above, and sharp protrusions 68c are formed at the corners of the regular hexagon. When viewed from the side of the ridgeline 68d, a local recess is formed between the two sharp projections 68c, and this recess is sometimes called a saddle. The two-dimensional size D68p of the recess 68p is approximately equal to the distance D0 between adjacent recesses 68p (D68p=D0), and the two-dimensional size D69p of the recess 69p is smaller than the distance D0 between adjacent recesses 69p. (D69p<D0). The two-dimensional size of the concave portion refers to the equivalent circle diameter of the concave portion when viewed from the normal direction of the surface. It is known that the distance D0 between adjacent recesses 69p corresponds to approximately twice the thickness tc of the barrier layer 69b and is approximately proportional to the voltage during anodization. The distance D0 between the adjacent recesses 68p, the distance D0 between the adjacent recesses 69p, and the two-dimensional size D68p of the recesses 68p are, for example, 10 nm or more and 1000 nm or less, and the two-dimensional size D69p of the recesses 69p is, for example, 5 nm. 500 nm or less. A depth Ddp of the recess 69p is, for example, 50 nm or more and 1000 nm or less.

Next, as shown in FIG. 6D, the remaining aluminum layer 68r on the transparent substrate 62 is anodized to form a porous alumina layer 64 having a plurality of recesses (pores) 64p2. Here, the remaining aluminum layer 68r is substantially completely anodized. This is because, in drop culture, it is preferable that the visible light transmittance of the porous alumina layer is high in order to observe the cells.

The shape of the porous alumina layer 64 will be described with reference to the plan view of FIG. 7B. The surface of the porous alumina layer 64 reflects the surface shape of the aluminum residual layer 68r. The surface of the porous alumina layer 64 has a plurality of recesses 64p1 and ridgelines 64d formed by the intersection of the side surfaces of the adjacent recesses 64p1. The concave portion 64p1 and the ridge line 64d reflect the concave portion 68p and the ridge line 68d, respectively, on the surface of the remaining aluminum layer 68r. The ridge line 64d is a side of a regular hexagon inscribed in a circle when the recess 64p1 is viewed from above, and sharp projections 64c are formed at the corners of the regular hexagon. When viewed from the side of the ridgeline 64d, a local recess is formed between the two pointed projections 64c, and this recess is sometimes called a saddle. The surface of the porous alumina layer 64 further has a plurality of micro-recesses 64p2 each formed within one of the plurality of recesses 64p1. The distance (center-to-center distance or pitch) between the recesses 64p1 and the micro recesses 64p2 is both D0, and the two-dimensional size Dp1 of the recesses 64p1 is approximately equal to the distance D0 between the recesses 64p1 (Dp1= D0), the two-dimensional size Dp2 of the micro recesses 64p2 is smaller than the distance D0 between adjacent micro recesses 64p2 (Dp2<D0).

The porous alumina layer 64 is formed, for example, by anodizing the surface of the remaining aluminum layer 68r in an acidic electrolyte. The electrolytic solution used in the step of forming the porous alumina layer 64 is, for example, an aqueous solution containing an acid selected from the group consisting of oxalic acid, tartaric acid, phosphoric acid, sulfuric acid, chromic acid, citric acid and malic acid. The anodizing step (first anodizing step) shown in FIG. 6B and the anodizing step (second anodizing step) shown in FIG. liquid) can be used.

Next, as shown in FIG. 6E, the opening of the micro recess 64p2 is enlarged by etching the porous alumina layer 64 by a predetermined amount by contacting it with an alumina etchant. That is, the two-dimensional size Dp2 of the micro recess 64p2 is increased. At this time, the shape of the ridgeline 64d and the pointed projection 64c also change (eg, become sharper as shown in FIG. 6E). The amount of etching (that is, the two-dimensional size and depth of the micro recesses 64p2) can be controlled by adjusting the type/concentration of the etchant and the etching time. As the etchant, for example, an aqueous solution of 10 mass % phosphoric acid, an organic acid such as formic acid, acetic acid or citric acid, an aqueous solution of sulfuric acid, or a mixed aqueous solution of chromic acid and phosphoric acid can be used. The first etching process (the process of removing the preliminary porous alumina layer 69) and the second etching process (the process of enlarging the opening of the micro recesses 64p2) are performed using, for example, the same etchant (the same kind, the same concentration, the same temperature etchant). The second etching step sharpens (sharpens) the cross-sectional shapes of the ridges 64d and the pointed projections 64c, thereby increasing the surface area and improving the water repellency of the porous alumina layer 64 surface. Note that the second etching process can be omitted.

In this way, a porous alumina layer 64 with a solid surface for use in the drop culture method is formed.

In the porous alumina layer 64 shown in FIG. 6E, the distance D0 between adjacent recesses 64p1, the distance D0 between adjacent micro recesses 64p2, and the two-dimensional size Dp1 of recesses 64p1 are, for example, 10 nm or more and 1000 nm or less. A two-dimensional size Dp2 of the recess 64p2 is, for example, 5 nm or more and 500 nm or less. The porous alumina layer 64 has a porous layer 64a (thickness ts) and a barrier layer (bottom of the micro recesses 64p2) 64b (thickness tb). The porous layer 64a has recesses 64p1 reflecting the recesses 68p of the surface of the aluminum residual layer 68r, and micro recesses (pores) 64p2 formed by anodizing the aluminum residual layer 68r. The thickness ts of the porous layer 64a corresponds to the sum of the depth Dd1 of the recess 64p1 and the depth Dd2 of the micro recess 64p2. The depth Dd1 of the concave portion 64p1 is, for example, 5 nm or more and 500 nm or less, and the depth Dd2 of the micro concave portion 64p2 is, for example, 50 nm or more and 1000 nm or less. The thickness ts of the porous layer 64a is, for example, 55 nm or more and 1500 nm or less. In addition, since the anodized film formed in the anodizing process may have a larger volume than the original aluminum, the thickness of the porous alumina layer 64 is larger than the thickness of the original aluminum layer (eg, 1000 nm). Sometimes.

Anodization and etching of the remaining aluminum layer 68r may be alternately performed multiple times. Either the anodizing step or the etching step may be completed. When forming an antireflection film using a porous alumina layer as a mold, it is preferable to end with an anodizing step. That is, since the bottom of the concave portion can be formed into a point, a mold capable of forming a convex portion with a sharp tip can be obtained. However, when the porous alumina layer is used for the drop culture method, the shape of the bottom of the recess does not affect the cell culture, so it is not necessary to finish with the anodizing step.

It is preferable to sterilize the surface of the porous alumina layer before performing the drop culture method using the surface of the porous alumina layer. The sterilization treatment may be a treatment that does not deform the surface shape of the porous alumina layer, that is, a treatment that does not seal the recesses of the porous alumina layer, such as UV sterilization, ethylene oxide gas (EOG) sterilization, γ-ray Sterilization can be performed.

Cell culture using a porous alumina layer is known (for example, Patent Document 10). It needs to be adjusted to be suitable for drop culture. For example, the surface of the porous alumina layer may be subjected to release treatment so that the surface of the porous alumina layer has appropriate water repellency. The release treatment can adjust the surface chemistry (eg, contact angle) of the porous alumina layer, as will be shown later. Moreover, the chemical properties of the surface of the porous alumina layer can be adjusted by applying a surface treatment agent to the surface of the porous alumina layer, not limited to the release agent.

The present inventors have found that one of the factors contributing to tissue reproducibility of spheroids obtained by the drop culture method is the degree (strength) of adhesion of cells to a solid surface. According to the study of the present inventors, depending on the cell type, when the adhesion of cells (pseudopodia of cells) to a solid surface is too weak, the tissue reproducibility of the obtained spheroids tends to be low. As shown in the experimental examples below, when the above porous alumina layer is used in the drop culture method, the type of release agent used for the release treatment of the porous alumina layer and the water repellency of the surface of the porous alumina layer affect the number of cells. Adhesion to solid surfaces was observed to vary.

(Experimental example 2)
Regarding the surface of the porous alumina layer used as the solid surface of the drop culture method, the result of examination of the porous alumina layer having appropriate cell adhesion will be described.

Using the method described with reference to FIGS. 6A-6E, 7A and 7B, specimen sample no. 101-105 were produced. Sample sample no. Each of 101 - 105 has a glass substrate 62 and a porous alumina layer 64 formed on the glass substrate 62 . As shown in Table 13, sample sample no. Sample Nos. 101 to 104 differ from each other only in the type of release agent used in the release treatment. 105 is a sample sample number. A porous alumina layer 64 was formed under conditions different from those of 101-104. In order to prepare each sample, an aluminum film 68 (thickness: 1000 nm) was formed on a glass substrate (manufactured by AGC Co., Ltd., product name: AN100, non-alkali glass, thickness 0.7 mm) 62 by sputtering. was used. The aluminum film was formed on a glass substrate using a sputtering apparatus (manufactured by Canon ANELVA, product name: C-3500). Using an aluminum target (purity 99.999%) manufactured by ULVAC, Inc., an aluminum film was obtained by intermittent deposition at a voltage of 500 V and a current of 400 A without heating. Intermittent deposition specifically means that an aluminum film having a desired thickness (for example, 1 μm) is formed by repeating the process of stopping the deposition at a certain thickness and resuming the deposition after a certain period of time has passed. means to obtain By intermittently forming a film without heating, it is possible to suppress an unnecessary rise in the surface temperature of the glass substrate, and as a result, it is possible to suppress variations in grain size on the surface of the aluminum film. can.

Table 13 shows the conditions of the manufacturing process (that is, the anodizing process, the etching process, and the release treatment process) of each sample after forming the aluminum film 68 on the glass substrate 62 . In Table 13, the first anodizing step is a step of forming the preliminary porous alumina layer 69 shown in FIG. 6B, and the first etching step substantially removes the preliminary porous alumina layer 69 shown in FIG. 6C. The second anodizing step is a step of anodizing the remaining aluminum layer 68r to form the porous alumina layer 64 shown in FIG. 6D, and the second etching step is and contacting the porous alumina layer 64 with an etchant, shown in FIG. 6E. The porous alumina layer 64 of each sample was obtained by anodizing and etching the remaining aluminum layer 68r once.

"Distance between electrodes" in Table 13 indicates the distance between the cathode and the sample (anode) in the anodizing step. "Conductive wire connection" in Table 13 is the method of connecting the sample (anode) and the anodizing power supply, and the surface of the aluminum film (that is, the surface 68s of the aluminum film 68 in the first anodizing process, the surface 68s of the aluminum film 68 in the second anodizing process, In the anodizing process, the method of connecting the surface of the aluminum residual layer 68r) and the conducting wire is shown. "Lead wire soldering" is a method of soldering a lead wire connected to an anodizing power supply to the surface of an aluminum film, and "aluminum foil + clip" is a method in which an aluminum foil is attached to a portion of the aluminum film surface. In this method, the foil is sandwiched between clips to make surface contact, and the aluminum foil and the anodizing power supply are connected.

The following fluoroalkylsilane (FAS) was used as a release agent. FAS3 (3,3,3-trifluoropropyltrimethoxysilane), FAS9 (nonafluorohexyltrimethoxysilane), FAS13 (tridecafluoro-1,1,2,2-tetrahydrooctyltrimethoxysilane) and FAS17 (hepta decafluoro-1,1,2,2-tetrahydrodecyltrimethoxysilane) was used. A fluoroalkylsilane has a perfluoroalkyl group (-(CF ₂ ) _p - group). The chemical formulas of FAS3, FAS9, FAS13 and FAS17 are CF ₃ —(CF ₂ ) _p —(CH ₂ ) ₂ Si(OCH ₃ ) ₃ . where p=0, p=3, p=5 and p=7 in FAS3, FAS9, FAS13 and FAS17, respectively.

The release treatment was performed by chemical vapor deposition. A beaker containing a fluoroalkylsilane (mold release agent) and a sample having a porous alumina layer 64 are placed in a sealed container made of Teflon (registered trademark) and sealed, and the sealed container is heated in an electric furnace. and held at 150° C. for 1 hour. In this manner, the fluoroalkylsilane was vaporized and chemical vapor deposition of the fluoroalkylsilane was performed on the sample (ie, on the surface of the porous alumina layer 64).

In order to examine the water repellency of the release agent, each release agent was applied to a flat surface, and the water repellency and oil repellency of the surface to which the release agent was applied were examined. Table 14 shows the results. The sample sample No. shown in Table 13 was used as a flat surface. The surface of the porous alumina layer obtained by performing the same treatment as the treatment conditions up to the anodization [first time] of 101 to 104 was used. That is, the porous alumina layer used here has the same structure as the preliminary porous alumina layer 69 shown in FIG. 6B. The surface of the preliminary porous alumina layer 69 has a plurality of recesses 69p, and the two-dimensional size D69p of the plurality of recesses 69p is smaller than the distance D0 between the adjacent recesses 69p (D69p = about 10 nm, D0 = about 200 nm). ), and the surface of the preliminary porous alumina layer 69 has a flat portion between the recesses 69p. Thus, the surface of the preliminary porous alumina layer 69 has higher flatness than the surface of the porous alumina layer 64, and can be regarded as a substantially flat surface in measuring the contact angle. The contact angle is measured by a general θ/2 method (half-angle Method: (θ/2 = arctan (h/r), θ: contact angle, r: radius of droplet, h: height of droplet s)) went. The amount of pure water and hexadecane used for contact angle measurement was 1 μL. Also, a droplet of 20 μL of pure water was used for the measurement of the sliding angle. The sliding angle is the inclination angle at which the droplet starts to slide downward when the surface on which the droplet adheres is tilted from the horizontal direction.

As can be seen from Table 14, both the contact angle to water and the contact angle to hexadecane of the flat surface to which the release agent is applied are the largest for FAS17, and decrease in the order of FAS13, FAS9, and FAS3. That is, the surface to which the release agent FAS3 is applied has the lowest water and oil repellency, and the surface to which the release agent FAS17 is applied has the highest water and oil repellency.

Figures 8A-8E show sample sample no. SEM images of 101 to 105 (after release treatment) are shown, respectively. For each of FIGS. 8A to 8E, the upper left and upper right are the surface SEM images (50000×) of the porous alumina layer, the upper left is the surface SEM image observed from the normal direction of the surface, and the upper right is the normal to the surface. It is a surface SEM image observed from an angle inclined by 45° from the direction. The lower left and lower right are cross-sectional SEM images of each sample, the lower right is a cross-sectional SEM image (30000x) observed from a direction perpendicular to the cross section, and the lower left is a 45° tilt from the direction perpendicular to the cross section. It is a cross-sectional SEM image (20000 times) observed from an angle.

Table 15 shows the result of obtaining the surface structure of the porous alumina layer 64 of each sample from the SEM image. The number of micro recesses 64p2 per area was counted by image processing using a surface SEM image. The average pitch of the micro recesses 64p2 was calculated from the number of holes. The thickness ts of the porous layer 64a was obtained from a cross-sectional SEM image. Since the thickness of the porous alumina layer 64 can expand to about 1.5 times the thickness of the original aluminum layer, the thickness ts of the porous layer 64a is greater than the thickness of the aluminum layer (here, 1000 nm). may also be large.

Table 15 also shows the results of measuring the contact angle and sliding angle in the same manner as in Experimental Example 1 as parameters that characterize the surface of the porous alumina layer 64 of each sample (the solid surface in the drop culture method). Here, a 1 μL droplet was used to measure the contact angle using pure water or hexadecane, and a 20 μL droplet of pure water was used to measure the sliding angle. The latter value was evaluated. The contact angle showed the same tendency as the flat surface evaluation results shown in Table 14, and the surface to which the release agent FAS3 was applied had the lowest water repellency and oil repellency, and the release agent FAS17 was applied. The water and oil repellency of the surface was the highest.

Table 15 also shows the results of measuring the transmittance of the porous alumina layer 64 of each sample at a wavelength of 550 nm. The transmittance at a wavelength of 550 nm was measured using an ultraviolet-visible-near-infrared spectrophotometer V7100 (manufactured by JASCO Corporation). The transmittance of the porous alumina layer 64 at a wavelength of 550 nm was measured using the sample No. 101 to 104 were all about 50%. As shown in Table 14, sample sample no. 101-104 were made using the same anodizing and etching steps. It can be seen that the transmittance of the porous alumina layer hardly changes depending on the type of release agent. The transmittance of the porous alumina layer at a wavelength of 550 nm is preferably 40% or more, for example.

Table 16 and FIG. 9 show sample sample nos. The results of culturing cells by the drop culture method using the surfaces of 101-104 are shown. Fig. 9 shows optical microscope images of spheroids obtained by the drop culture method (upper left: sample sample No. 101, upper right: sample sample No. 102, lower left: sample sample No. 103, lower right: sample sample No. 104). is.

The drop culture method was performed under the same conditions as in Experimental Example 1 (HepG2 cells, 1×10 ⁵ cells/mL, medium droplet volume 25 μL).

For the "spheroid size" in Table 16, the largest one among the area circle equivalent diameters when the spheroid (cell cluster) is viewed from above was evaluated in three stages of +/++/+++. +: Equivalent circle diameter is less than 70 μm, ++: Equivalent circle diameter is 70 μm or more and less than 200 μm, +++: Equivalent circle diameter is 200 μm or more.

"Spheroid height" in Table 16 evaluated the height of spheroids (cell clumps) according to the degree of blackness (darkness of color) when viewed from above. The blacker (the darker the color) when viewed from above, the higher the evaluation. It was evaluated in three grades of +/++/+++.

"Spheroid uniformity" in Table 16 evaluated the variation in the area of the spheroids (cell clumps) when viewed from above in three grades of +/++/+++.

In Table 16, "isolated cell quantity", the number of isolated cells was evaluated in 3 grades of +/++/+++. Here, in contrast to spheroids (cell clumps), which are composed of multiple cells gathered (bonded), cells that do not gather (unbond) and exist separately are defined as isolated cells. . +: 100 or more isolated cells, ++: 30 or more and less than 100 isolated cells, +++: less than 30 isolated cells. In the upper left and upper right optical microscope images of FIG. 9, examples of spheroids (cell clumps) are indicated by solid line circles, and isolated cells are indicated by dotted line circles.

"Pseudopodia growth" in Table 16 evaluated the length of filamentous pseudopodia extending from spheroids (cell clusters) in three grades of +/++/+++. X indicates that no pseudopodia were observed.

"Adhesion to cells on solid surface" in Table 16 is the result of evaluating the degree (strength) of adhesion of cells to the solid surface as follows. Every time the medium was replaced (every 24 hours), the culture vessel was intentionally shaken (the culture vessel was shaken). After culturing for 3 days, the degree of adhesion of the cells to the culture vessel and the presence or absence of pseudopodia of the cells were visually observed. The degree of adhesion of cells to the solid surface (culture vessel) was evaluated according to the following criteria and expressed as the degree of adhesion of the solid surface to cells.
x: Pseudopodia were not observed, and cell migration was observed when observing the state in which the culture vessel was vibrated. That is, the shaking of the medium (droplets) by vibration caused the cells to move away from the solid surface.
Δ: Partial pseudopodia were observed, but cell migration was observed when observing the state in which the culture vessel was vibrated.
◯: Pseudopodia were observed, but cell migration was observed when observing the state in which the culture vessel was vibrated. However, the cells did not migrate when the container was moved lightly without forcible vibration.
A: Pseudopodia were observed, and cell migration was not observed when observing the state in which the culture vessel was vibrated.

Sample Sample No. using release agent FAS3. In 101, it was observed that the pseudopodia of the cells were growing and that the pseudopodia of the cells were adhered to the solid surface with moderate strength. On the other hand, sample sample Nos. using release agents FAS9, FAS13 and FAS17, respectively. In 102-104, the cell pseudopodia had not grown and the cell pseudopodia had not adhered to the solid surface with reasonable strength. Sample sample no. It is believed that the adhesion of cells to solid surfaces is weak due to the surface of 102-104 being too water repellent. Sample sample no. The size uniformity of the spheroids obtained with sample No. It was higher than 102-104. From the viewpoint of spheroid size uniformity, the fluorine-based release agent includes, for example, a fluorine compound having a perfluoroalkyl group (—(CF ₂ ) _p — group, p is an integer of 0 or more and 2 or less). It can be seen that it is preferable to use a fluorine-based release agent. By using such a fluorine-based release agent, the solid surface can have appropriate water repellency and oil repellency. The contact angle of the solid surface with water is, for example, 108° or more and 128° or less. The contact angle of the solid surface to hexadecane is, for example, 57° or more and 85° or less.

Sample sample no. 102-104, the homogeneity of the size of the spheroids obtained is as good as sample sample no. Although lower than 101, there was a tendency to obtain larger spheroids. Depending on the application, it may be preferable to obtain spheroids of uniform size, and in other cases (for example, non-adherent cells (hematopoietic cells)), larger spheroids are preferable. Depending on the application of the spheroids, the solid surface used for the drop culture method can be selected and the chemical properties (eg contact angle) of the solid surface can be adjusted.

As described above, according to the embodiments of the present invention, a three-dimensional culture method capable of obtaining spheroids with higher workability or mass productivity and/or higher tissue reproducibility than conventional three-dimensional culture methods. is provided.

A three-dimensional culture structure provided with a solid surface having a plurality of protrusions with a height of 10 nm or more and 1 mm or less, such as the synthetic polymer membrane provided with a surface having a moth-eye structure exemplified in the examples, is suitable for the drop culture method. used for Such a three-dimensional culture structure can be obtained, for example, by attaching the above synthetic polymer film to the inner bottom surface of a petri dish. That is, the three-dimensional culture structure can be provided as a container such as a petri dish.

Furthermore, by performing drop culture using such a three-dimensional culture structure (eg, vessel), it is possible to produce a three-dimensional culture structure (eg, vessel) having spheroids on its surface with higher tissue reproducibility than in the past. . A three-dimensional culture structure having such spheroids on its surface is suitably used for research and development of drug discovery and regenerative medicine.

A three-dimensional cell culture method according to an embodiment of the present invention can be widely used in drug discovery, regenerative medicine, and the like.

10S solid surface 10Sp convex 12C cell 14M medium 16D droplet

Claims (16)

providing a cell suspension comprising cells and medium;
providing a solid surface including a surface of a porous alumina layer having a plurality of first recesses;
depositing droplets of the cell suspension onto the surface of the porous alumina layer ;
and culturing the cells in the droplet with the direction of gravity acting on the droplet toward the surface of the porous alumina layer . The surface of the porous alumina layer includes any one of the plurality of first recesses, first ridgelines formed by intersections of side surfaces of the plurality of adjacent first recesses, and the plurality of first recesses, respectively. The three-dimensional culture method according to claim 1, having a plurality of micro-recesses formed therein. The step of forming the porous alumina layer includes
Step (a) of forming a preliminary porous alumina layer by partially anodizing an aluminum film on a transparent substrate;
a step (b) of substantially completely removing the preliminary porous alumina layer after step (a) by contacting the preliminary porous alumina layer with an etchant;
After the step (b), a step (c) of anodizing the remaining aluminum layer of the aluminum film that has not been anodized,
The surface of the aluminum residual layer has a plurality of second recesses and second ridgelines formed by the intersection of side surfaces of the plurality of adjacent second recesses,
The plurality of first recesses reflects the plurality of second recesses, the first ridgeline reflects the second ridgeline,
3. The three-dimensional culture method according to claim 2, wherein said step (c) comprises forming said plurality of micro-recesses by anodizing said aluminum residual layer. 4. The three-dimensional culture method according to claim 3, wherein the anodizing step performed in step (c) is performed using the same electrolytic solution as the anodizing step performed in step (a). The step of forming the porous alumina layer includes
The three-dimensional culture according to claim 3 or 4, further comprising a step (d) of enlarging the plurality of micro-recesses by contacting the porous alumina layer with an etchant after the step (c). law. The three-dimensional culture method according to claim 5, wherein the etchant used in step (d) is the same as the etchant used in step (b). 7. The three-dimensional culture method according to any one of claims 3 to 6, wherein step (c) comprises substantially completely anodizing the remaining aluminum film. The three-dimensional culture method according to any one of claims 1 to 7, wherein the transmittance of the porous alumina layer at a wavelength of 550 nm is 40% or more. The three-dimensional culture method according to any one of claims 1 to 8, wherein the surface of the porous alumina layer is subjected to mold release treatment with a fluorine-based mold release agent. The three-dimensional culture method according to claim 9, wherein the fluorine-based release agent contains a fluorine compound having a perfluoroalkyl group. 10. The three-dimensional culture method according to claim 9, wherein the fluorine-based release agent contains a fluorine compound having a perfluoroalkyl group (-(CF ₂ ) _p - group, p is an integer of 0 or more and 2 or less). The three-dimensional culture method according to any one of claims 1 to 11, wherein the surface of the porous alumina layer has a water contact angle of 108° or more and less than 127° . The three-dimensional culture method according to any one of claims 1 to 12, wherein the contact angle of said surface of said porous alumina layer to hexadecane is 57° or more and 85° or less. 14. The three-dimensional culture method according to any one of claims 1 to 13, wherein the cells are adhered to the surface of the porous alumina layer while culturing the cells in the droplet. A three-dimensional culture structure having a solid surface for use in the three-dimensional culture method according to any one of claims 1 to 14 . A three-dimensional culture structure having the solid surface is prepared, and a three-dimensional culture structure having spheroids cultured using the three-dimensional culture method according to any one of claims 1 to 14 on the solid surface is manufactured. Method.

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