JP7303962B2 - Culture vessel and method for producing cell-carrier complex - Google Patents

Description

TECHNICAL FIELD The present invention relates to a culture vessel and a method for producing a cell-carrier complex.

Patent Document 1 discloses a process of seeding mesenchymal cells and epithelial cells on a micro-intaglio plate consisting of regularly arranged micro-concavities and culturing them while supplying oxygen to form a hair follicle primordium. A method for producing aggregates of regenerated follicle primordia is described, comprising:

Patent Document 2 discloses a method for producing a regenerated organ primordium for transplantation having a guide, wherein the first cell aggregate is substantially composed of mesenchymal cells and the epithelial cells. a second cell aggregate and culturing inside a support carrier to prepare a regenerated organ primordium; and a step of inserting a guide into the regenerated organ primordium. there is

WO2017/073625 WO2012/108069

However, in the method described in Patent Document 2, a regenerated organ primordium is first prepared, and then a guide is inserted into the regenerated organ primordium, so that the operation requires skill. Moreover, when preparing a large amount of regenerated organ primordia, the operation of inserting a guide into each of the regenerated organ primordia was inevitably complicated.

The present invention has been made in view of the above problems, and one of the objects thereof is to provide a culture vessel and a method for producing a cell-carrier complex that can easily produce a cell-carrier complex. To be one.

A culture vessel according to one embodiment of the present invention for solving the above problems is a culture vessel including wells for culturing cells, wherein the culture vessel is fixed to the culture vessel, and at least a portion thereof is for culturing the cells. A fibrous carrier disposed within the well is included therein. INDUSTRIAL APPLICABILITY According to the present invention, a culture vessel is provided in which a cell-carrier complex can be easily produced.

In the culture vessel, one carrier may be arranged in one well. In the culture vessel, the carrier may be fixed to the inner surface of the well. In this case, part of the carrier may extend outside the well.

Moreover, in the culture vessel, the carrier may be fixed to a portion of the culture vessel outside the well. In this case, it may further include a support that is arranged above the well during the culture of the cells, and the carrier may be fixed to the support. Moreover, in these cases, the support portion may be detachably provided.

A method for producing a cell-carrier complex according to one embodiment of the present invention for solving the above problems comprises preparing a culture vessel containing wells, and fixing at least part of a fiber-like carrier to the culture vessel. and culturing the cells to form a cell-carrier complex comprising the cells adhered to the carrier. INDUSTRIAL APPLICABILITY According to the present invention, a method for producing a cell-carrier complex is provided, which enables the simple production of a cell-carrier complex.

The method may further comprise recovering the cell-carrier complex by separating the carrier from the culture vessel. In the method, the cells may be cultured to form the cell-carrier complex containing cell aggregates adhered to the carrier. In the method, the culture vessel may be any culture vessel described above.

INDUSTRIAL APPLICABILITY According to the present invention, a culture vessel and a method for producing a cell-carrier complex are provided, which allow easy production of a cell-carrier complex.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory diagram showing an oblique appearance of an example of a culture vessel according to the present embodiment; It is an explanatory view showing the culture container shown in FIG. 1 in a plan view. FIG. 3 is an explanatory view showing a cross section of the culture vessel cut along line III-III in FIG. 2; FIG. 4 is an explanatory diagram showing another example of the cross-sectional shape of wells included in the culture vessel according to the present embodiment; FIG. 5 is an explanatory diagram showing still another example of the cross-sectional shape of wells included in the culture vessel according to the present embodiment. FIG. 5 is an explanatory diagram showing still another example of the cross-sectional shape of wells included in the culture vessel according to the present embodiment. FIG. 5 is an explanatory diagram showing still another example of the cross-sectional shape of wells included in the culture vessel according to the present embodiment. FIG. 5 is an explanatory diagram showing still another example of the cross-sectional shape of wells included in the culture vessel according to the present embodiment. FIG. 4 is an explanatory diagram showing another example of a fiber-like carrier contained in the culture vessel according to the present embodiment; FIG. 4 is an explanatory diagram showing still another example of a fiber-like carrier contained in the culture vessel according to the present embodiment; FIG. 4 is an explanatory diagram showing still another example of a fiber-like carrier contained in the culture vessel according to the present embodiment; FIG. 3 is an explanatory diagram showing an example of a fiber-like carrier and a supporting portion included in the culture vessel according to the present embodiment; FIG. 4 is an explanatory diagram showing another example of a fiber-like carrier and a supporting portion included in the culture vessel according to the present embodiment; FIG. 10 is an explanatory diagram showing still another example of the fiber-like carrier and the supporting part contained in the culture vessel according to the present embodiment. FIG. 10 is an explanatory diagram showing still another example of the fiber-like carrier and the supporting part contained in the culture vessel according to the present embodiment. FIG. 10 is an explanatory diagram showing still another example of the fiber-like carrier and the supporting part contained in the culture vessel according to the present embodiment. FIG. 10 is an explanatory diagram showing still another example of the fiber-like carrier and the supporting part contained in the culture vessel according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing a part of the steps included in one example of the method for producing a cell-carrier complex according to the present embodiment. FIG. 3 is an explanatory diagram schematically showing another part of the steps included in one example of the method for producing a cell-carrier complex according to the present embodiment. FIG. 4 is an explanatory diagram schematically showing still another part of the steps included in one example of the method for producing a cell-carrier complex according to the present embodiment. FIG. 4 is an explanatory diagram schematically showing still another part of the steps included in one example of the method for producing a cell-carrier complex according to the present embodiment. FIG. 4 is an explanatory diagram schematically showing still another part of the steps included in one example of the method for producing a cell-carrier complex according to the present embodiment. FIG. 4 is an explanatory diagram schematically showing still another part of the steps included in one example of the method for producing a cell-carrier complex according to the present embodiment. FIG. 4 is an explanatory view schematically showing another example of part of the steps included in one example of the method for producing a cell-carrier complex according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing a part of the steps included in another example of the method for producing a cell-carrier complex according to this embodiment. FIG. 4 is an explanatory diagram schematically showing another part of the steps included in another example of the method for producing a cell-carrier complex according to this embodiment. FIG. 4 is an explanatory diagram schematically showing still another part of the steps included in another example of the method for producing a cell-carrier complex according to this embodiment. FIG. 4 is an explanatory diagram schematically showing still another part of the steps included in another example of the method for producing a cell-carrier complex according to this embodiment. FIG. 4 is an explanatory diagram schematically showing still another part of the steps included in another example of the method for producing a cell-carrier complex according to this embodiment. FIG. 4 is an explanatory diagram schematically showing still another part of the steps included in another example of the method for producing a cell-carrier complex according to this embodiment. FIG. 4 is an explanatory view schematically showing another example of part of the steps included in another example of the method for producing a cell-carrier complex according to this embodiment. FIG. 4 is an explanatory diagram schematically showing a part of the steps included in still another example of the method for producing a cell-carrier complex according to this embodiment. FIG. 4 is an explanatory view schematically showing another part of the steps included in still another example of the method for producing a cell-carrier complex according to this embodiment. FIG. 4 is an explanatory diagram schematically showing still another part of the steps included in still another example of the method for producing a cell-carrier complex according to this embodiment. FIG. 4 is an explanatory view showing phase-contrast micrographs of cell-carrier complexes produced in Examples according to the present embodiment.

An embodiment of the present invention will be described below. Note that the present invention is not limited to this embodiment. In the present specification, the terms “upper” and “lower” respectively refer to the upper and lower directions when cells are statically cultured using a culture vessel. Further, the same reference numerals may be assigned to corresponding elements in multiple drawings, and detailed description thereof may be omitted.

First, the culture vessel according to this embodiment will be described. FIG. 1 shows an oblique appearance of an example of a culture vessel 1 according to this embodiment. FIG. 2 shows the culture vessel 1 shown in FIG. 1 in plan view. FIG. 3 shows a cross section of the culture vessel 1 taken along line III--III shown in FIG.

The culture vessel 1 includes a well 20 for culturing cells, and a fibrous carrier (hereinafter referred to as " (referred to as "fiber carrier") 30;

Well 20 is used for culturing cells therein. A space 21 is formed in the well 20 to hold cells and culture medium. A space 21 of well 20 is surrounded by an inner surface 22 . The inner surface 22 of well 20 includes a bottom surface 22a and side surfaces 22b.

Here, the bottom surface 22a of the well 20 is a portion of the inner surface 22 that is visible in plan view (see FIG. 2). Cells seeded in well 20 settle by gravity in the culture medium and deposit on bottom surface 22a. Well 20 has opening 23 above bottom surface 22a. For example, seeding of cells into well 20 is through opening 23 .

The inner surface 22 (particularly the bottom surface 22a) of the well 20 is preferably cell non-adhesive. When the inner surface 22 of the well 20 is non-adherent to cells, the cells do not adhere to the inner surface 22 during the culture of the cells in the well 20, but float in the culture medium or are removed from the culture medium by pipetting or the like. is easily detached from the inner surface 22 by flowing the The shape of the cells cultured on the cell non-adhesive inner surface 22 is maintained approximately spherical.

On the other hand, when the inner surface 22 of the well 20 is cell-adhesive, the cells adhere to the inner surface 22 during the culture of the cells in the well 20, and even if the culture medium is flowed by pipetting or the like, the inner surface 22 is not easily detached. Cells cultured on the cell-adhesive inner surface 22 adhere to the inner surface 22 and extend, and their shape becomes flatter than when they are floating in the culture medium.

The shape of the bottom surface 22a of the well 20 and the opening 23 is not particularly limited as long as the effects of the present invention can be obtained, and may be circular, elliptical, polygonal, or any other shape. The area of ^the bottom ^surface 22a of the well 20 is not particularly limited as long as the effects of the present invention ^can be obtained. 100000 μm ² or more is particularly preferable. The area of the bottom surface 22a of the well 20 may be, for example, 1000 mm ² or less, preferably 100 mm ² or less, more preferably 50 mm ² or less, and particularly preferably 20 mm ² or less. .

The area of the bottom surface 22a of the well 20 may be specified by arbitrarily combining one of the above lower limits and one of the above upper limits. Specifically, the area of the bottom surface 22a of the well 20 may be, for example, 100 μm ² or more and 1000 mm 2 or less, preferably 1000 μm ² or more and 100 mm ² or less, and 10000 μm ^{2 or} more and 50 mm ² or less. More preferably, it is 100000 μm ² or more and 20 mm ² or less.

The depth of the well 20 (the distance between the lowest part of the bottom surface 22a and the upper end of the well 20 (for example, the surface 11 of the substrate 10 in which the opening 23 is formed)) is particularly limited as long as the effects of the present invention can be obtained. However, for example, it may be 100 μm or more, preferably 150 μm or more, more preferably 200 μm or more, and particularly preferably 300 μm or more. Also, the depth of the well 20 may be, for example, 30 mm or less, preferably 20 mm or less, more preferably 10 mm or less, and particularly preferably 5 mm or less.

The depth of the well 20 may be specified by arbitrarily combining one of the above lower limits and one of the above upper limits. Specifically, the depth of the well 20 may be, for example, 100 μm or more and 30 mm or less, preferably 150 μm or more and 20 mm or less, more preferably 200 μm or more and 10 mm or less, and 300 μm or more. , 5 mm or less.

Although the number of wells 20 included in the culture vessel 1 is not particularly limited, the culture vessel 1 preferably includes a plurality of wells 20 . Furthermore, the culture vessel 1 preferably includes a plurality of regularly arranged wells 20, as shown in FIGS. Specifically, the culture vessel 1 includes, for example, a plurality of wells 20 arranged at regular intervals. The culture vessel 1 also includes, for example, multiple wells 20 forming multiple columns and multiple rows.

In the examples shown in FIGS. 1 to 3, the culture vessel 1 includes a substrate 10 and wells 20 formed in the substrate 10. FIG. Specifically, the surface 11 of the substrate 10 is formed with an opening 23 of the well 20 . The substrate 10 is not particularly limited as long as it is a plate-like member. For example, the shape of the substrate 10 (the shape visually recognized in plan view as in FIG. 2) is not particularly limited, and may be rectangular, circular, elliptical, or any other shape. The material constituting the substrate 10 is not particularly limited, and an arbitrary material such as an organic polymer such as resin, an inorganic material such as glass, or a metal may be used, but an organic polymer such as resin is preferably used.

The fiber carrier 30 has a fibrous shape and is a carrier to which cells can adhere at least partly. The material constituting the fiber carrier 30 is not particularly limited as long as the effects of the present invention can be obtained, and any material such as an organic material, an inorganic material, or a metal can be used, for example. As the fiber carrier 30, for example, artificial fibers or natural fibers are preferably used.

Examples of artificial fibers include artificial organic fibers such as synthetic resin fibers, semi-synthetic fibers, regenerated fibers, artificial inorganic fibers such as glass fibers, and metal fibers such as stainless steel fibers. Examples of natural fibers include animal-derived fibers, plant-derived fibers, and mineral-derived fibers. As the animal-derived fiber, for example, human hair or non-human animal hair (including non-human mammal hair, bird hair, and insect-derived fiber such as silk fiber) is used. As plant-derived fibers, for example, cotton fibers or hemp fibers are used. A biodegradable fiber may be used as the fiber carrier 30 .

The fiber carrier 30 preferably has flexibility, and particularly preferably has flexibility similar to animal hair (especially human hair). The shape of the fiber carrier 30 is not particularly limited as long as the effects of the present invention can be obtained, but preferably has a shape similar to animal hair (particularly human hair), for example. The cross-sectional shape of the fiber carrier 30 is not particularly limited as long as the effects of the present invention can be obtained. .

Each fiber carrier 30 is preferably a single fibrous carrier that exists independently. That is, the fiber carrier 30 is not woven with other fibers, for example. Also, the fiber carrier 30 does not constitute, for example, a sheet-like cloth (for example, woven cloth and non-woven cloth). Moreover, the fiber carrier 30 may be a hollow body (for example, hollow fiber) or a solid body, but is preferably a solid body. It is preferable that the fiber carrier 30 has a cell-adhesive surface at least at the part with which cells come into contact.

The cross-sectional area of the fiber carrier 30 ⁽ the cross-sectional area perpendicular to the longitudinal direction of the fiber carrier 30) is not particularly limited as long as the effects of the present invention can be obtained. , is preferably 300 μm ² or more, more preferably 500 μm ² or more, and particularly preferably 1000 μm ² or more. The cross-sectional area of the fiber carrier 30 may be, for example, 1 mm ² or less, preferably 100000 μm ² or less, more preferably 50000 μm ² or less, and particularly preferably 30000 μm ² or less. .

The cross-sectional area of the fiber carrier 30 may be specified by any combination of one of the above lower limits and one of the above upper limits. Specifically, the cross-sectional area of the fiber carrier 30 may be, for example, 100 μm ² or more and 1 mm ² or less, preferably 300 μm ² or more and 100000 μm ² or less, and 500 μm ² or more and 50000 μm ² or less. 1000 μm ² or more and 30000 μm ² or less is particularly preferable.

The length of the fiber carrier 30 is not particularly limited as long as the effects of the present invention can be obtained. More preferably, it is particularly preferably 400 μm or more. Also, the length of the fiber carrier 30 may be, for example, 30 mm or less, preferably 20 mm or less, more preferably 15 mm or less, and particularly preferably 10 mm or less.

The length of the fiber carrier 30 may be specified by any combination of one of the above lower limits and one of the above upper limits. Specifically, the length of the fiber carrier 30 may be, for example, 100 μm or more and 30 mm or less, preferably 200 μm or more and 20 mm or less, more preferably 300 μm or more and 15 mm or less, and 400 μm. Above all, it is particularly preferable to be 10 mm or less.

The number of fiber carriers 30 arranged in one well 20 in the culture vessel 1 is not particularly limited as long as the effects of the present invention can be obtained. is preferably arranged. In this case, one fiber carrier 30 is placed in each well 20, as shown in FIGS.

A fiber carrier 30 is fixed to the culture vessel 1 . That is, a portion of the fiber carrier 30 that is a fiber-like member is fixed to the culture vessel 1 . In addition, a part of the fiber carrier 30 is fixed to the culture vessel 1 means that a part of the fiber carrier 30 and a part of the culture vessel 1 to which the part of the fiber carrier 30 is fixed. It means that the relative positional relationship is fixed.

The portion of the fiber carrier 30 fixed to the culture vessel 1 is not particularly limited as long as the effects of the present invention can be obtained. For example, as shown in FIGS. It is fixed to the culture container 1 .

The portion of the culture vessel 1 to which the fiber carrier 30 is fixed is not particularly limited as long as the effects of the present invention can be obtained. good. That is, in the example shown in FIGS. 1 to 3, the fiber carrier 30 has a portion (specifically, one end 31) fixed to the inner surface 22 (specifically, the bottom surface 22a) of the well 20. It is

The fiber carrier 30 may have free ends. 1 to 3, the fiber carrier 30 has one end 31 (fixed end) fixed to the culture vessel 1 and the other end 32 which is a free end. .

A portion of the fiber carrier 30 may extend outside the well 20 when the fiber carrier 30 is secured to the inner surface 22 of the well 20 . That is, in the example shown in FIG. 3 , the fiber carrier 30 has one end 31 fixed to the inner surface 22 of the well 20 and the other end 32 (free end) extending from the opening 23 of the well 20 . It extends upwards. In other words, the free end 32 of the fiber carrier 30 protrudes from the surface 11 of the substrate 10 in which the well 20 is formed. Thus, the length of fiber carrier 30 may be greater than the depth of well 20 .

At least a portion of the fiber carrier 30 is placed within the well 20 during cell culture, as described below. In the example shown in FIGS. 1-3, as described above, the free end (end 32) of fiber carrier 30 is positioned outside well 20, resulting in a portion of fiber carrier 30 being positioned within well 20. there is One end 31 of the fiber carrier 30 fixed to the bottom surface 22a of the well 20 is arranged near the bottom surface 22a.

The purpose of arranging the fiber carrier 30 in the well 20 is to allow the cells cultured in the well 20 to adhere to the fiber carrier 30, as will be described later. In this respect, it is preferable to arrange at least part of the fiber carrier 30 in contact with the bottom surface 22a of the well 20. However, as described later, even if the fiber carrier 30 does not contact the bottom surface 22a By arranging at least part of the fiber carrier 30 in the vicinity of the bottom surface 22 a on which the cells are present during cell culture, the cells can be adhered to at least part of the fiber carrier 30 .

Specifically, for example, during cell culture, at least part of the fiber carrier 30 is arranged within a range of 500 μm or less from at least part of the bottom surface 22a of the well 20 . The distance between at least part of the fiber carrier 30 and at least part of the bottom surface 22a of the well 20 during the cell culture is preferably 100 μm or less, more preferably 10 μm or less, and 1 μm or less. is particularly preferred.

In the culture vessel 1, at least a part of the fiber carrier 30 may be placed inside the well 20 during cell culture. That is, the fiber carrier 30 does not have to be placed in the well 20 before starting cell culture or after completing cell culture. Of course, in the culture vessel 1, at least part of the fiber carrier 30 may be arranged in the well 20 regardless of whether cells are being cultured.

In the example shown in FIG. 3, the well 20 of the culture vessel 1 is a so-called flat-bottom well having a flat (horizontal) bottom surface 22a, but the shape of the well 20 is not limited to this. It may have a contoured inner surface 22 . That is, the well 20 may be, for example, a so-called U-bottomed well having a curved U-shaped bottom surface 22a as shown in FIG. 4A, or a tapered bottom surface 22a as shown in FIG. 4B. Alternatively, it may have a V-shaped bottom surface 22a (inner surface 22) as shown in FIG. 4C. Also, the cross-sectional shape of the well 20 may be another shape.

The culture vessel 1 may further include a cavity 40 that accommodates the well 20, as shown in FIG. 4D. A space 41 for holding a culture medium is formed in the cavity 40 . A well 20 is formed on the surface 11 forming the bottom surface of the cavity 40 . Retaining culture medium in cavity 40 necessarily retains culture medium in wells 20 as well. That is, the wells 20 are entirely submerged in the culture medium.

In the example shown in FIG. 4D, the well 20 and the cavity 40 are formed in the same substrate 10. However, as shown in FIG. A cavity 40 may be formed in the substrate 45 that has been formed. That is, in this case, the substrate 10 with the wells 20 formed therein is placed in the cavity 40 formed in the substrate 45 that is molded separately from the substrate 10 .

In the example shown in FIG. 3, the fiber carrier 30 is fixed by inserting its one end 31 into the bottom surface 22a of the well 20 (specifically, a part of the substrate 10 forming the bottom surface 22a). However, the manner in which the fiber carrier 30 is fixed to the inner surface 22 of the well 20 is not limited to this.

That is, for example, the fiber carrier 30 may be partially fixed to a fixing layer 24 arranged in the well 20 and made of a material different from that of the substrate 10, as shown in FIG. 5A. In this case, part of the fiber carrier 30 (one end 31 in the example shown in FIG. 5A) is embedded in the fixing layer 24 to fix the fiber carrier 30 to the culture vessel 1 . When the fiber carrier 30 is fixed to the fixing layer 24 in the well 20, the upper surface of the fixing layer 24 becomes the bottom surface 22a of the well 20, as shown in FIG. 5A.

Specifically, for example, first, a fixing layer 24 made of gel is formed in the well 20, and then one end 31 of the fiber carrier 30 is pierced into the fixing layer 24 to obtain the fiber carrier 30. is fixed to the fixing layer 24 .

Alternatively, for example, first, a raw material liquid before gelation is put into the well 20 to form a raw material liquid layer having a gelling ability, and then one end 31 of the fiber carrier 30 is inserted into the raw material liquid layer, Thereafter, the fiber carrier 30 is fixed to the fixing layer 24 by gelling the raw material liquid layer to form the fixing layer 24 .

The gel that constitutes the fixing layer 24 is not particularly limited as long as it can fix a part of the fiber carrier 30. For example, a synthetic polymer gel or a naturally derived polymer gel is preferably used. Moreover, a cell non-adhesive gel is preferably used for the fixing layer 24 . Hydrophilic polymer gels are preferably used as non-cell-adhesive gels, such as agarose gels, alginic acid gels, and synthetic polymer gels (e.g., gels containing polyacrylamide, polyvinyl alcohol, methylcellulose, polyethylene oxide, etc.). One or more hydrophilic polymer gels selected from the group consisting of are preferably used.

In the example shown in FIG. 3, the fiber carrier 30 is a member molded separately from the substrate 10, but is not limited to this. That is, the fiber carrier 30 may be molded integrally with the substrate 10 as shown in FIG. 5B. In this case, it can be said that the fiber carrier 30 is molded integrally with the well 20 (specifically, the bottom surface 22a in the example shown in FIG. 5B). Therefore, the fiber carrier 30 and the substrate 10 are made of the same material. On the other hand, when the fiber carrier 30 is molded separately from the substrate 10, the material forming the fiber carrier 30 may be different from or the same as the material forming the substrate 10. FIG.

In the example shown in FIG. 3, the fiber carrier 30 partially extends outside the well 20, but is not limited to this. That is, the fiber carrier 30 may be placed entirely within the well 20, as shown in FIG. 5C. Specifically, in the example shown in FIG. 5C, the fiber carrier 30 has one end 31 fixed to the inner surface 22 (specifically, the bottom surface 22a) of the well 20, and the other end 32 is a free end. is placed in the space 21 of the well 20 as a.

Also, the fiber carrier 30 may be fixed to a portion of the culture vessel 1 outside the well 20 . In this case, the portion of the culture vessel 1 to which the fiber carrier 30 is fixed is not particularly limited as long as it is a portion other than the well 20. 10), or a member other than the substrate 10. FIG.

That is, the culture vessel 1 may further include a support that is arranged above the wells 20 during cell culture, and the fiber carrier 30 may be fixed to the support. FIG. 6A shows an example of this case.

In the example shown in FIG. 6A, the culture vessel 1 includes a support 50 arranged above the wells 20 . A portion of the fiber carrier 30 is fixed to the support portion 50 . Another part of the fiber carrier 30 is arranged inside the well 20 .

That is, the fiber carrier 30 is suspended from a support 50 arranged above the well 20 . A portion of the fiber carrier 30 then extends downward into the well 20 . Specifically, one end portion 31 of the fiber carrier 30 is fixed to the support portion 50 . The other end 32 of the fiber carrier 30 is arranged as a free end inside the well 20 (specifically, near the bottom surface 22a of the well 20).

In addition, the culture vessel 1 may further include a positioning part that fixes the position of the support part 50 in the culture vessel 1 during cell culture. FIG. 6A shows an example of this case. In the example shown in FIG. 6A , the culture vessel 1 includes a positioning portion 51 that fixes the position of the support portion 50 .

The positioning portion 51 is fixed to the substrate 10 and connected to the support portion 50 . The positioning portion 51 fixes the position of the support portion 50 relative to the substrate 10 . As a result, the relative position of the portion of the fiber carrier 30 fixed to the support 50 (one end 31 of the fiber carrier 30 in the example shown in FIG. 6A) relative to the well 20 or the substrate 10 is also fixed.

In the example shown in FIG. 6A , the positioning portion 51 is fixed to the substrate 10 by fitting the convex portion 52 formed on the positioning portion 51 into the concave portion 12 formed on the surface 11 of the substrate 10 . However, the method of fixing the positioning portion 51 to the substrate 10 is not limited to the concave-convex structure, and any method such as an engaging structure including hooks or the like, or an adhesive structure using an adhesive may be used.

The support part 50 may be arranged directly above the well 20 as shown in FIG. 6A , but is not limited to this and may be arranged at a position other than directly above the well 20 . FIG. 6B shows an example of this case. In the example shown in FIG. 6B, the support portion 50 is positioned above the well 20 and above the surface 11 of the substrate 10 on which the well 20 is formed (specifically, between a pair of adjacent wells 20). position in between).

The support part 50 preferably does not completely close the openings 23 of the wells 20 during cell culture. That is, during cell culture, in the culture container 1, the culture medium is placed in the well 20 through the opening 23 of the well 20, or the culture medium is discharged from the well 20. Preferably, the opening 23 is maintained.

Specifically, the support part 50 preferably does not cover the entire opening 23 of the well 20 in plan view of the culture vessel 1 (see FIG. 2). In this regard, in both examples shown in FIGS. 6A and 6B , the support part 50 does not cover the entire opening 23 of the well 20 in plan view of the culture container 1 .

That is, in the example shown in FIG. 6A , the support part 50 covers only part of the opening 23 of the well 20 in plan view of the culture container 1 . In addition, in the example shown in FIG. 6B, the support part 50 does not cover the opening 23 of the well 20 at all when the culture container 1 is viewed from above.

In the culture vessel 1 having the support portion 50, the fiber carrier 30 may extend from the inside of the well 20 to the outside of the well 20 as shown in FIGS. 6A and 6B, but is not limited to this. It may be located entirely within well 20 . FIG. 6C shows an example of this case.

In the example shown in FIG. 6C, one end 31 of the fiber carrier 20 is fixed to the support 50 on the same plane as the upper end surface of the well 20, that is, the surface 11 of the substrate 10 on which the well 20 is formed, The fiber carrier 20 is suspended from the support 50 .

Therefore, the entire fiber carrier 30 is placed in the well 20 including one end 31 fixed to the support 50 . Moreover, in the example shown in FIG. 6C , the positioning portion 51 is arranged at a position between adjacent wells 20 .

Note that the support portion 50 whose cross section is shown in FIG. 6C may be a lid portion that covers the entire opening 23 of the well 20. It is good also as blocking only a part. In this regard, the support part 50 may have a structure that blocks only a part of the opening 23 of the well 20 in plan view of the culture container 1, as shown in FIG. 6D.

When the culture container 1 includes a cavity 40 that accommodates the well 20, the support section 50 may be arranged above the cavity 40 as shown in FIG. 6E. In the example shown in FIG. 6E, the positioning portion 51 is fixed to the surface 43 in which the cavity 40 is formed. Moreover, when the culture container 1 includes the cavity 40, the positioning part 51 may be fixed to the surface 11 in which the wells 20 are formed, as shown in FIG. 6F. Moreover, when the culture container 1 includes the cavity 40 , the support section 50 may be arranged inside the cavity 40 .

When the culture vessel 1 includes the support portion 50 arranged above the cavity 40, the fiber carrier 30 fixed to the support portion 50 is pushed into the well from outside the cavity 40 as shown in FIGS. 6E and 6F. Extends to within 20.

Also, the support portion 50 may be provided detachably. In this case, the support part 50 is formed separately from the substrate 10 of the culture vessel 1, as shown in FIGS. 6A to 6F, for example. The support part 50 is preferably fixed to the substrate 10 via the positioning part 51 during cell culture.

On the other hand, the support part 50 can be detached from the substrate 10 before the cell culture is started or after the cell culture is finished. Even if the support part 50 is detachably provided, the support part 50 may remain fixed to the substrate 10 before starting cell culture or after completing cell culture. Moreover, the support part 50 may be temporarily detached during cell culture.

In the examples shown in FIGS. 6A, 6B, 6E, and 6F, the positioning portion 51 is formed by fitting the convex portion 52 formed in the positioning portion 51 into the concave portion 12 formed in the surface 11 of the substrate 10 . Although the support part 50 is detachably provided via the Any method can be used, such as the adhesive structure used.

Next, a method for producing a cell-carrier complex according to the present embodiment (hereinafter referred to as "this method") will be described. Figures 7A-7F schematically illustrate the steps involved in one example of this method.

The method includes preparing a culture vessel 1 including wells 20, culturing cells 60 in the wells 20 in which at least a portion of the fiber carrier 30 fixed to the culture vessel 1 is arranged, and culturing the cells 60 to form a cell-carrier complex 100 comprising the cells 60 adhered to the fiber carrier 30 .

In this method, first, a culture vessel 1 including wells 20 is prepared. The culture vessel 1 is preferably the culture vessel described above. That is, as shown in FIG. 7A, a culture vessel 1 is prepared in which a fiber carrier 30, at least a part of which is arranged in a well 20, is fixed.

Cells 60 are then cultured in wells 20 in which at least a portion of fiber carrier 30 is placed. That is, as shown in FIG. 7B, the culture of the cells 60 is started by seeding the cells 60 in the wells 20 of the culture vessel 1 . Specifically, a cell suspension containing cells 60 dispersed in culture medium 80 is placed in wells 20 .

The cells 60 cultured in this method are not particularly limited as long as they are living cells derived from animals. The animal may be a human or a non-human animal (animal other than human), but it is preferable to use human cells. Non-human animals are not particularly limited, but are preferably non-human vertebrates (vertebrates other than humans). Non-human vertebrates are not particularly limited, but preferably non-human mammals. Non-human mammals include, but are not limited to, primates (e.g., monkeys), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), carnivores (e.g., dogs, cats), or animals. Hoofed animals (eg, pigs, cows, horses, goats, sheep).

The cells may be differentiated cells or undifferentiated cells (stem cells). Stem cells may be totipotent stem cells, pluripotent stem cells, or tissue stem cells. Specifically, stem cells may be, for example, induced pluripotent (iPS) stem cells, embryonic stem (ES) cells, or embryonic germ (EG) cells. . A differentiated cell is not particularly limited as long as it is a cell having a differentiated function, and may be, for example, a cell collected from a living body (or may be a cell cultured after being collected from a living body). Alternatively, cells induced from stem cells in vitro may be used.

The cells may be cells derived from tissues in vivo. In vivo tissue is not particularly limited, but for example, hair follicle tissue, skin tissue, liver tissue, heart tissue, kidney tissue, nerve tissue, bone tissue, cartilage tissue, bone marrow tissue, lung tissue, glandular tissue, periodontal tissue It may be tissue or blood.

Preferably, the cells are adherent cells. Adherent cells are cells that exist in vivo in a state of adhering to other cells and/or extracellular matrix. As the cells, only one type of cells may be used, or two or more types of cells may be used in combination. When two or more types of cells are combined, these cells are co-cultured.

Combinations of two or more types of cells are not particularly limited as long as they are combinations of different interacting cells. A combination of different cells is, for example, a combination of cells in which one or more different cells selected from the group consisting of differentiation function, proliferation, and cell surface markers are used.

A combination of two or more types of cells is preferably a combination of cells that interact in vivo. In this case, the combination of cells may be a combination of interacting cells in the same tissue in vivo. In vivo tissue is not particularly limited, but for example, hair follicle tissue, glandular tissue, skin tissue, liver tissue, heart tissue, kidney tissue, nerve tissue, bone tissue, cartilage tissue, bone marrow tissue, lung tissue, periodontal tissue It may be tissue or blood.

A combination of two or more types of cells may be a combination of cells derived from the same animal or a combination of cells derived from different animals. That is, the two or more types of cells may be either human cells (cells derived from humans) or non-human animal cells (cells derived from animals other than humans), One may be a human cell and the other may be a non-human animal cell. However, it is preferable to use two or more types of human cells.

A combination of two or more types of cells is preferably, for example, a combination of epithelial cells and mesenchymal cells. The combination of epithelial cells and mesenchymal cells is not particularly limited. A combination of active epithelial cells and mesenchymal cells is preferred.

Epithelial cells and/or mesenchymal cells are cells collected from living hair follicle tissue or gland tissue (e.g., one or more selected from the group consisting of sweat glands, sebaceous glands, lacrimal glands, and salivary glands) (the hair follicle It may be a cell cultured after being collected from a tissue), or a cell induced from a stem cell in vitro.

Specifically, the epithelial cells are derived from outer root sheath outermost layer cells of the bulge region of the hair follicle tissue, epithelial cells derived from the base of the hair matrix, or stem cells (e.g., iPS cells, ES cells, or EG cells). It may be a hair follicle epithelial system cell. Also, the epithelial cells may be epithelial stem cells.

The mesenchymal cells are hair follicle mesenchymal cells derived from dermal papilla cells, dermal root sheath cells, nascent skin mesenchymal cells, or stem cells (e.g., iPS cells, ES cells, or EG cells). It can be a certain thing.

Epithelial cells may have hair growth-related properties (eg, expression of hair growth-related genes and/or formation of hair follicle primordium by co-culture with mesenchymal cells). The epithelial cells may be cells derived from hair follicle tissue (e.g., the outermost layer of the outer root sheath of the bulge region of the hair follicle tissue and/or the base of the hair matrix), or cells derived from skin tissue. They may also be cells derived ex vivo from stem cells (eg, iPS stem cells, ES cells, or EG cells). Epithelial cells may be primary cells collected from a living body, or pre-cultured cells (eg, subcultured cells and/or established cells). Epithelial cells are identified, for example, as cells that express cytokeratin. The epithelial cells are preferably epithelial stem cells. Epithelial stem cells are identified, for example, as cells expressing cytokeratin 15 and/or CD34.

Mesenchymal cells may have hair growth-related properties (eg, expression of hair growth-related genes and/or formation of hair follicle primordium by co-culture with epithelial cells). The mesenchymal cells may be cells derived from adult hair follicle tissue (e.g., dermal papilla and/or hair bulb root sheath), and skin tissue (fetal, juvenile, or adult skin tissue). ) derived from stem cells (e.g., induced pluripotent (iPS) stem cells, embryonic stem (ES) cells, or embryonic germ (EG) cells) in vitro. cells may be used. Mesenchymal cells may be primary cells collected from a living body, or pre-cultured cells (eg, subcultured cells and/or established cells). Mesenchymal cells are identified as cells expressing, for example, Versican and ALP (alkaline phosphatase). Specifically, as the mesenchymal cells, dermal papilla cells and/or hair bulb hair root sheath cells are preferably used. Dermal papilla cells and hair bulb root sheath cells express Versican and ALP.

The culture medium 80 is not particularly limited as long as it is a liquid containing components for maintaining the survival of the cells 60 therein. is used.

The cells 60 are cultured by holding the cells 60 in a culture medium in the well 20 at a temperature suitable for culturing the cells 60 and in a gas phase having a composition and humidity suitable for culturing the cells 60. conduct. Moreover, in this method, in order to obtain the cells 60 adhered to the fiber carrier 30 , the culture is performed while at least part of the seeded cells 60 are in contact with the fiber carrier 30 .

After the cells 60 are seeded in the wells 20, the culture vessel 1 is allowed to stand still, so that the cells 60 are placed on the bottom surface 22a of the wells 20 by gravity in the culture medium 80 in the wells 20, as shown in FIG. 7C. settle on top.

As a result, some of the cells 60 settle around the fiber carrier 30 (specifically, around one end 31 of the fiber carrier 30 fixed to the bottom surface 22a of the well 20), and the fiber carrier 30 is held in contact with

The density of the cells 60 cultured in the well 20 is not particularly limited as long as the effect of the present invention is obtained. For example, as shown in FIG. In some cases, it is preferable to seed and culture the cells 60 at such a density that multiple cell layers are stacked.

By continuing the culture while at least part of the seeded cells 60 are in contact with the fiber carrier 30, at least part of the cells 61 adhere to the fiber carrier 30 as shown in FIG. 7D.

That is, when a part of the surface of the fiber carrier 30 that is in contact with the cells 60 is previously treated to impart cell adhesiveness (for example, collagen coating treatment), at least some of the cells 61 are Adheres to the cell-adhesive surface.

Further, even if the treatment for imparting cell adhesiveness is not performed, the adherent cells 61 are, for example, components such as proteins contained in the culture medium 80, and / or the cells 61 are in the culture medium 80 It adheres to the surface of the fiber carrier 30 via a component such as an extracellular matrix that is secreted into the membrane.

By culturing the cells 60 in this manner, a cell-carrier complex 100 including the fiber carrier 30 and the cells 61 adhered to the fiber carrier 30 is formed as shown in FIG. 7D.

Further, by continuing the culture even after some of the cells 61 adhere to the fiber carrier 30, as shown in FIG. can be formed.

That is, by continuing the culture with some cells 61 adhering to the fiber carrier 30 and other cells 60 being in contact with the said some cells 61, the said some cells 61 and the other cells 60 , and the formation of bonds between the other cells 60, the cells 60 spontaneously aggregate.

As a result, a cell aggregate 70 including cells 61 adhered to the fiber carrier 30 and surrounding a portion of the fiber carrier 30 is formed. In other words, a cell aggregate 70 is formed in which a portion of the fiber carrier 30 is arranged.

Thus, in this method, the cell-carrier complex 100 can be produced. According to this method, the cell-carrier complex 100 can be easily produced by culturing the cells 60 using the culture vessel 1 in which the fiber carrier 30 is fixed.

In addition, as shown in FIGS. 1 and 2, by culturing cells 60 in a plurality of wells 20 in which fiber carriers 30 are arranged, a large amount of cell-carrier complexes 100 can be efficiently produced. can.

In addition, the method may further include recovering the cell-carrier complex 100 by separating the fiber carrier 30 from the culture vessel 1 after the cell-carrier complex 100 is formed.

That is, since the fiber carrier 30 is a fibrous member, it can be easily separated from the culture vessel 1. As a result, the cell-carrier complex 100 containing the fiber carrier 30 separated from the culture vessel 1 can be recovered from well 20. In this case, the recovered cell-carrier complex 100 contains the fiber carrier 30, which is the fiber piece separated from the culture vessel 1. FIG.

Specifically, for example, as shown in FIGS. 7A to 7E, when the fiber carrier 30 is inserted into and fixed to the bottom surface 22a of the well 20, as shown in FIG. 7F, the fiber carrier 30 is The cell-carrier complex 100 can be recovered by pulling it out from the bottom surface 22a. In the example shown in FIG. 7F, the bottom surface 22a of the well 20 after collecting the cell-carrier complex 100 exposes the recess 13 into which one end 31 of the fiber carrier 30 was inserted.

Further, for example, as shown in FIG. 5A, when the fiber carrier 30 is fixed to the fixing layer 24 made of gel, the cell-carrier complex 100 can be obtained by pulling out the fiber carrier 30 from the fixing layer 24. can be recovered.

Further, for example, as shown in FIG. 5B, when the fiber carrier 30 is molded integrally with the substrate 10, the portion (one end portion) of the fiber carrier 30 fixed to the bottom surface 22a of the well 20 31) is cut with an instrument such as a scalpel or scissors, the cell-carrier complex 100 can be recovered.

In the culture vessel 1, when the fiber carrier 30 is fixed to the inner surface 22 such as the bottom surface 22a of the well 20, part of the fiber carrier 30 extends outside the well 20. It is preferable because the operation for separating from the culture vessel 1 is facilitated.

7A to 7E, part of the fiber carrier 30 extends out of the culture medium 80 in the well 20, as shown in FIG. 5C. It is preferable because it facilitates the operation for separating the fiber carrier 30 from the culture vessel 1, including the case where it is arranged inside.

FIG. 7G shows cells 60 held in wells 20 when culture vessel 1 includes cavities 40 that accommodate wells 20 . In this case, the cavity 40 holds the culture medium 80 and the cells 60 are cultured in the wells 20 placed in the culture medium 80 . In the example shown in FIG. 7G, a portion of the fiber carrier 30 (specifically, one end 31) is fixed to the inner surface 22 (specifically, the bottom surface 22a) of the well 20, and the other portion is (Specifically, the other end 32, which is a free end) extends out of the culture medium.

Figures 8A-8F schematically illustrate the steps involved in another example of the method. In the example shown in FIGS. 8A-8F, the culture vessel 1 includes a support 50 positioned above the wells 20 and to which the fiber carrier 30 is fixed.

In this method, first, as shown in FIG. 8A, culture vessel 1 is prepared in which at least part of fiber carrier 30 having one end 31 fixed to support 50 is placed in well 20 . In this culture vessel 1 , the free end 32 of the fiber carrier 30 suspended from the support 50 is arranged near the bottom surface 22 a of the well 20 .

Cells 60 are then cultured in wells 20 in which at least a portion of fiber carrier 30 is placed. That is, as shown in FIG. 8B, the cells 60 dispersed in the culture medium 80 are seeded in the wells 20 of the culture vessel 1 to start culturing the cells 60 .

After that, by leaving the culture vessel 1 still, the cells 60 settle on the bottom surface 22a of the well 20 by gravity in the culture medium in the well 20, as shown in FIG. 8C. As a result, some of the cells 60 settle around the fiber carrier 30 (specifically, around the other end 32 (free end) of the fiber carrier 30 arranged near the bottom surface 22a of the well 20). , is held in contact with the fiber carrier 30 .

By continuing the culture with at least some of the cells 60 in contact with the fiber carrier 30, some of the cells 61 adhere to the fiber carrier 30 as shown in FIG. 8D. That is, by culturing the cells 60 in the well 20 in which the fiber carrier 30 is placed, the cell-carrier complex 100 including the fiber carrier 30 and the cells 61 adhered to the fiber carrier 30 is formed.

Furthermore, by continuing to culture the cells 60 around the fiber carrier 30, a cell-carrier complex 100 including cell aggregates 70 adhered to the fiber carrier 30 is formed, as shown in FIG. 8E.

After the formation of the cell-carrier complex 100, as shown in FIG. 8F, the fiber carrier 30 is separated from the culture vessel 1 (specifically, the support section 50), thereby separating the cell-carrier complex 100. can be recovered. Specifically, in the example shown in FIG. 8F, the fiber carrier 30 fixed to the support 50 is separated from the support 50 by cutting the vicinity of one end 31 of the fiber carrier 30 with an instrument such as a scalpel or scissors. A cell-carrier complex 100 including the fiber carrier 30 and a cell aggregate 70 formed around the other end 32 of the fiber carrier 30 can be recovered.

FIG. 8G shows cells 60 held in the wells 20 when the culture vessel 1 including the support portion 50 includes the cavities 40 that accommodate the wells 20 . In this case, the cavity 40 holds the culture medium 80 and the cells 60 are cultured in the wells 20 placed in the culture medium 80 . In the example shown in FIG. 8G, a part (specifically, one end 31) of the fiber carrier 30 is fixed to the support 50 arranged above the cavity 40, and the other part (specifically, the The other end 32 ), which is essentially a free end, extends into the well 20 . Note that the support part 50 may be arranged inside the cavity 40 and outside the culture medium 80 held in the cavity 40 .

In the examples shown in FIGS. 7A to 7F and FIGS. 8A to 8F, the cells 60 were seeded in the well 20 in which at least part of the fiber carrier 30 was previously placed. The cells 60 may be seeded in the wells 20 in which the cells are not arranged, and then the fiber carriers 30 may be arranged in the wells 20 . 9A-9C schematically show some of the steps involved in one example of the method in this case.

In this method, as shown in FIG. 9A, a culture vessel 1 is prepared in which no fiber carrier 30 is arranged in the well 20 . Cells 60 are then cultured in wells 20 in which at least a portion of fiber carrier 30 is placed. First, as shown in FIG. 9B, cells 60 dispersed in a culture medium 80 are seeded in wells 20 in which fiber carriers 30 are not arranged.

Next, as shown in FIG. 9C, at least part of the fiber carrier 30 is placed in the wells 20 in which the cells 60 have been seeded in advance, thereby starting the culture of the cells 60 . That is, after seeding the cells 60, by placing the support 50 to which the fiber carrier 30 is fixed in the culture vessel 1, a part of the fiber carrier 30 (specifically, the free end of the fiber carrier 30) The portion 32) is immersed in the culture medium 80 in the well 20 and placed near the bottom surface 22a. The support 50 is installed by fixing the positioning portion 51 connected to the support 50 to the substrate 10 .

The cell-carrier complex 100 produced by this method is applied to various uses by utilizing the feature of including the fiber carrier 30 . That is, for example, when the cell-carrier complex 100 contains cells for transplantation into a living body, the cell-carrier complex 100 is used as a graft. A living body into which the cell-carrier complex 100 is transplanted may be a human or a non-human animal, but is preferably a human.

Specifically, when the cell-carrier complex 100 contains cells derived from hair follicle tissue or cells derived from glandular tissue, the cell-carrier complex 100 is transplanted into a living body to regenerate hair follicle tissue or glandular tissue. can do. In the implanted cell-carrier complex 100, the fiber carrier 30 serves, for example, as a guide for the growth and/or migration of the cells contained in the cell-carrier complex 100.

When the cell-carrier complex 100 produced by this method is transplanted into a living body and hair is grown from the cell-carrier complex 100, the living body is not particularly limited, and may be a human or a non-human animal. There may be. The cell-carrier complex 100 is preferably transplanted into the skin of the living organism. Implantation into the skin may be, for example, subcutaneous implantation or intradermal implantation.

The production of the cell-carrier complex 100 and its transplantation into a living body may be for medical use or research use. That is, for example, for the treatment or prevention of a disease associated with hair loss, the cell-carrier complex 100 is produced for the purpose of transplantation into a human patient suffering from or likely to suffer from the disease, and the cell-carrier complex Body 100 may be implanted into the human patient.

Diseases associated with hair loss are not particularly limited, but for example, androgenetic alopecia (AGA), female androgenetic alopecia (FAGA), postpartum alopecia, diffuse alopecia, seborrhea Composed of alopecia areata, alopecia pityriasis, traction alopecia, metabolic alopecia, alopecia areata compressive, alopecia areata, alopecia areata, alopecia nervosa, trichotillomania, alopecia generalis, and symptomatic alopecia It may be one or more selected from the group.

In addition, for example, in order to search for substances that can be used in the treatment or prevention of diseases associated with hair loss and/or to search for substances involved in the mechanism of the disease, the cell-carrier complex 100 is produced, or the cells - The carrier complex 100 may be implanted in a non-human animal.

After transplanting the cell-carrier complex 100 into the living body, the fiber carrier 30 contained in the cell-carrier complex 100 is removed. That is, for example, by pulling out the fiber carrier 30 extending from the implanted cell-carrier complex 100 with forceps, the fiber carrier 30 can be removed. Moreover, when the fiber carrier 30 is composed of a biodegradable material, the fiber carrier 30 is decomposed by hydrolysis or the like and removed after implantation.

Next, specific examples according to this embodiment will be described.

[Collection of epithelial cells and mesenchymal cells]
The back skin tissue was collected from C57BL/6 mouse fetuses of 18 days of gestational age, and the method reported by Nakao et al. was performed at 4° C. for 1 hour under shaking conditions of 30 rpm to separate the epithelial layer and the mesenchymal layer of the skin tissue. Thereafter, the epithelial layer was treated with 100 U/mL collagenase for 1 hour and 20 minutes, and then treated with trypsin for 10 minutes to isolate epithelial cells. The mesenchymal layer was treated with 100 U/mL collagenase for 1 hour and 20 minutes to isolate mesenchymal cells.

[Manufacture of culture vessel]
A culture vessel containing a plurality of regularly arranged wells was manufactured. That is, first, using CAD software (V Carve Pro 6.5), a multiwell pattern to be produced was designed by computer. Next, a cutting machine was used to cut the olefinic resin substrate according to the designed pattern, thereby producing a concave mold having the pattern. An epoxy resin (Crystal Lysine, manufactured by Nisshin Resin Co., Ltd.) was poured into this mold, cured for one day, and then released from the mold to form a convex mold having the above designed pattern. The formed convex mold was fixed to the bottom of a 24-well plate, polydimethylsiloxane (PDMS) was poured into it and solidified. A multiwell culture vessel having a diameter of 1 mm and a depth of 1 mm was prepared.

On the other hand, a stainless fiber (outer diameter 100 μm, length 10 mm) was prepared as a fiber carrier. Collagen coating was applied to the stainless fiber in order to enhance cell adhesion on the surface of the stainless fiber.

Then, one end of the stainless fiber coated with collagen was pierced into the bottom surface made of PDMS of the well of the multiwell culture vessel, thereby fixing the stainless fiber to the culture vessel. One stainless steel fiber was arranged in one well. Thus, a culture vessel containing wells in which stainless steel fibers as fiber carriers were arranged was obtained.

[Production of cell-carrier complex]
The epithelial cells and the relevant epithelial cells are added so that the cell density of the epithelial cells and the mesenchymal cells is 4×10 ³ cells/well (that is, the total cell density is 8×10 ³ cells/well). It was mixed with mesenchymal cells and seeded in each well of the culture vessel produced as described above.

As the culture medium, a mixed medium prepared by mixing a mesenchymal cell culture medium (DMEM+10% FBS+1% penicillin/streptomycin) and HuMedia-KG2 at a volume ratio of 1:1 was used. After that, cell culture was performed for 3 days in each well.

[result]
Three days of cell culture resulted in the formation of cell aggregates adhered to the surface of the fiber carrier. After culturing the cells for 3 days, the fiber carrier was pulled out from the bottom of the well to collect a cell-carrier complex composed of the fiber carrier and cell aggregates adhered to the fiber carrier.

FIG. 10 shows a phase-contrast micrograph of the recovered cell-carrier complex. The scale bar in FIG. 10 indicates 200 μm. As shown in FIG. 10, a cell-carrier complex containing cell aggregates attached to a portion of the fiber carrier was obtained.

Claims (8)

A culture vessel comprising wells for culturing cells,
A fiber-like carrier fixed to the culture vessel, at least a part of which is arranged in the well during culture of the cells,
The carrier has one end that is fixed to the inner surface of the well and the other end that is a free end that extends upward, or is fixed to a portion of the culture vessel outside the well. ,
culture vessel. 2. The culture vessel according to claim 1, wherein one carrier is placed in one well. The carrier has the one end fixed to the inner surface of the well,
3. The culture vessel according to claim 1, wherein part of said carrier extends outside said well . further comprising a support positioned above the well during culturing of the cells;
The carrier is fixed to the support,
The culture vessel according to claim 1 or 2 . The culture vessel according to claim 4 , wherein the support part is detachably provided. Preparing the culture vessel according to any one of claims 1 to 5 ,
culturing cells in the well in which at least part of the fibrous carrier fixed to the culture vessel is placed; and
Culturing the cells to form a cell-carrier complex comprising the cells adhered to the carrier;
A method for producing a cell-carrier complex, comprising: 7. The method for producing a cell-carrier complex according to claim 6 , further comprising recovering the cell-carrier complex by separating the carrier from the culture vessel. 8. The method for producing a cell-carrier complex according to claim 6 , wherein the cell-carrier complex containing the cell aggregate adhered to the carrier is formed by culturing the cells.

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