JP7403734B2 - Fiber carrier-cell-containing gel complex, method for producing the same, and kit for producing fiber carrier-cell-containing gel complex - Google Patents

Description

The present invention relates to a fiber carrier-cell-containing gel complex, a method for producing the same, and a kit for producing the fiber carrier-cell-containing gel complex.

Patent Document 1 discloses a method for producing a regenerating organ primordium for transplantation having a guide, the first cell aggregate consisting essentially of mesenchymal cells and the first cell aggregate consisting essentially of epithelial cells. A manufacturing method is described that includes the steps of: preparing a regenerating organ primordium by bringing the second cell aggregate into close contact with the second cell aggregate and culturing it inside a support carrier; and inserting a guide into the regenerating organ primordium. There is.

Patent Document 2 describes a method for producing a regenerated hair follicle primordium for transplantation in which the color of the hair that is generated after transplantation is controlled, including a step of preparing a first cell aggregate containing mesenchymal cells, and a step of preparing a first cell aggregate containing mesenchymal cells; a step of preparing a second cell aggregate containing cells; a step of preparing a cell aggregate containing pigment stem cells; a step of preparing a cell aggregate containing pigment stem cells; and subsequently, at least one of the first cell aggregate and the second cell aggregate is bonded to the cell aggregate containing the pigment stem cells. Regenerated hair for transplantation, comprising the step of bringing the first cell aggregate and the second cell aggregate into close contact with each other and culturing them inside a support member, and further comprising the step of inserting a guide into the regenerated hair follicle primordium. A method for producing envelope primordia is described.

International Publication No. 2012/108069 International Publication No. 2012/115079

However, in the methods described in Patent Document 1 and Patent Document 2, a regenerating organ primordium is first prepared, and then a guide is inserted into the regenerating organ primordium, so the operation requires skill. Furthermore, when preparing a large number of regenerative organ primordia, a guide is inserted into each of the large number of regenerative organ primordia, so a large number of guides must be inserted and a large number of regenerative organ primordia with guides inserted must be handled. In this case, the operation had to be complicated.

The present invention has been made in view of the above problems, and provides an easy-to-handle fiber carrier-cell-containing gel complex, a method for simply producing a fiber carrier-cell-containing gel complex, and a fiber carrier-cell-containing gel complex. One of the purposes is to provide a kit for easily producing a gel complex containing the present invention.

A method for producing a fiber carrier-cell-containing gel composite according to an embodiment of the present invention to solve the above problems includes (1) preparing a base material and a fiber carrier disposed on the surface of the base material. (2) forming droplets on the surface of the substrate in a gas phase that adhere to the fiber carrier and include hydrogel polymers and dispersed cells; (3) gelling the droplets on the surface of the substrate during the phase to obtain a fiber carrier-cell-containing gel composite comprising the fiber carrier and a cell-containing gel body attached to the fiber carrier; ,including. According to the present invention, a method for easily producing a fiber carrier-cell-containing gel composite is provided.

The method may further include detaching the cell-containing gel body of the fiber carrier-cell-containing gel composite from the surface of the base material. Furthermore, the method may further include culturing the cells within the cell-containing gel body of the fiber carrier-cell-containing gel complex. In this case, the cells may be cultured in the cell-containing gel body to form aggregates of the cells.

In the method, in (2), the hydrogel polymer and the dispersed cells are attached to one of the fiber carriers at a distance from each other on the surface of the base material in the gas phase. forming a plurality of droplets containing the same, and in step (3), gelling the droplets on the surface of the substrate in a gas phase, and forming a plurality of droplets with the one fiber carrier at a distance from each other. The fiber carrier-cell-containing gel composite may be obtained, which includes a plurality of the cell-containing gel bodies attached to the one fiber carrier. In this case, the method includes cutting the one fiber carrier of the fiber carrier-cell-containing hydrogel composite to remove one fiber carrier piece and the cell-containing gel body attached to the one fiber carrier piece. The method may further include obtaining a fiber carrier-cell-containing gel composite piece comprising:

In the method, in (1), a plurality of the fiber carriers arranged on the surface of the base material are prepared, and (2) and (3) are performed for each of the plurality of fiber carriers. It may also be a thing. Further, in the method, in (1) above, the fiber carrier is prepared, a part of which is fixed to the support member, and another part of which is arranged on the surface of the base material, and the fiber carrier is fixed to the support member. The above-mentioned (2) and (3) may be carried out for the fiber carrier.

In the method, (2) comprises (2-1) first cells attached to the fiber carrier and dispersed with a first hydrogel polymer on the surface of the base material in a gas phase; (2-2) in a gas phase, on the surface of the substrate, adhering to the fiber carrier and being dispersed with a second hydrogel polymer; forming a second droplet containing a second cell and connected to the first droplet to obtain a connected droplet attached to the fiber carrier, in (3) above, A fiber carrier-cell-containing gel composite comprising the fiber carrier and the cell-containing gel body attached to the fiber carrier by gelling the droplet linkage on the surface of the base material in a gas phase. It is also possible to obtain . In this case, the combination of the first cell and the second cell may be a combination of an epithelial cell and a mesenchymal cell.

A fiber carrier-cell-containing gel composite according to an embodiment of the present invention for solving the above problems includes one fiber carrier and one fiber, each containing a hydrogel polymer and a cell, spaced apart from each other. a plurality of cell-containing gel bodies attached to a carrier. According to the present invention, a fiber carrier-cell-containing gel complex that is easy to handle is provided.

In the complex, each of the plurality of cell-containing gel bodies may include aggregates of the cells. In addition, in the complex, each of the plurality of cell-containing gel bodies includes a first gel part containing a first hydrogel polymer and a first cell, a first gel part containing a first hydrogel polymer and a second cell, and a first gel part containing a first hydrogel polymer and a first cell. A second gel portion containing cells may also be included. Furthermore, in the complex, the combination of the first cell and the second cell may be a combination of an epithelial cell and a mesenchymal cell.

A kit for producing a fiber carrier-cell-containing gel composite according to an embodiment of the present invention for solving the above problems comprises: a fiber carrier to which a cell-containing gel body containing a hydrogel polymer and cells is attached; a support member to which is fixed. According to the present invention, a kit for easily producing a fiber carrier-cell-containing gel complex is provided.

The kit further includes a base material on the surface of which the fiber carrier fixed to the support member is placed in a gas phase, and on which a cell-containing gel body attached to the fiber carrier is formed. Good too.

According to the present invention, there is provided a fiber carrier-cell-containing gel complex that is easy to handle, a method for simply producing a fiber carrier-cell-containing gel complex, and a method for easily producing a fiber carrier-cell-containing gel complex. kit is provided.

FIG. 2 is an explanatory diagram schematically showing some of the main steps included in an example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing other parts of the main steps included in an example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing still another part of the main steps included in an example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing still another part of the main steps included in an example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing still another part of the main steps included in an example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing still another part of the main steps included in an example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing still another part of the main steps included in an example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing still another part of the main steps included in an example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing, in plan view, some of the main steps included in another example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing, in plan view, other parts of the main steps included in another example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing, in plan view, still another part of the main steps included in another example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing, in plan view, still another part of the main steps included in another example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing, in plan view, still another part of the main steps included in another example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing, in plan view, still another part of the main steps included in another example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing, in plan view, still another part of the main steps included in another example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. FIG. 2 is an explanatory diagram schematically showing, in plan view, still another part of the main steps included in another example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment. 2A is an explanatory diagram schematically showing a cross section taken along line III-A shown in FIG. 2A. FIG. FIG. 2B is an explanatory diagram schematically showing a cross section taken along line III-B shown in FIG. 2B. FIG. 2C is an explanatory diagram schematically showing a cross section taken along line III-C shown in FIG. 2C. FIG. 2D is an explanatory diagram schematically showing a cross section taken along the line III-D shown in FIG. 2D. FIG. 2E is an explanatory diagram schematically showing a cross section taken along line III-E shown in FIG. 2E. FIG. 2F is an explanatory diagram schematically showing a cross section taken along line III-F shown in FIG. 2F. 2G is an explanatory diagram schematically showing a cross section taken along line III-G shown in FIG. 2G. FIG. FIG. 2H is an explanatory diagram schematically showing a cross section taken along line III-H shown in FIG. 2H. FIG. 2 is an explanatory diagram showing a phase contrast micrograph of a complex on the first day of culture in Example 1 according to the present embodiment. FIG. 2 is an explanatory diagram showing a phase contrast micrograph of the complex on the third day of culture in Example 1 according to the present embodiment. FIG. 2 is an explanatory diagram showing a fluorescence micrograph of the complex on the first day of culture in Example 1 according to the present embodiment. FIG. 2 is an explanatory diagram showing a fluorescence micrograph of the complex on the third day of culture in Example 1 according to the present embodiment. FIG. 2 is an explanatory diagram showing an example of the results of observing hair growth three weeks after implantation of the fiber carrier-cell-containing gel complex in Example 2 according to the present embodiment. FIG. 3 is an explanatory diagram showing an example of the results of observing hair growth 3 weeks after transplantation of only a cell-containing gel body without a fiber carrier in Example 2 according to the present embodiment. FIG. 7 is an explanatory diagram showing an example of the result of manufacturing a composite including a fiber carrier fixed to a support member and a cell-containing gel body attached to the fiber carrier in Example 3 according to the present embodiment. FIG. 7 is an explanatory diagram showing an optical micrograph of the complex on the first day of culture in Example 3 according to the present embodiment. FIG. 7 is an explanatory diagram showing an optical micrograph of a complex on the third day of culture in Example 3 according to the present embodiment. FIG. 7 is an explanatory diagram showing a phase contrast micrograph of a complex immediately after the start of culture in Example 3 according to the present embodiment. FIG. 3 is an explanatory diagram showing a phase contrast micrograph of a complex on the third day of culture in Example 3 according to the present embodiment. FIG. 3 is an explanatory diagram showing a fluorescence micrograph of a complex immediately after the start of culture in Example 3 according to the present embodiment. FIG. 7 is an explanatory diagram showing a fluorescence micrograph of the complex on the third day of culture in Example 3 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.

1A to 1H schematically show the main steps included in an example of the method for producing a fiber carrier-cell-containing gel composite according to the present embodiment (hereinafter referred to as "the present method"). The method includes (1) providing a substrate 100 and a fiber carrier 10 disposed on a surface 110 of the substrate 100 (FIG. 1A); (2) disposing the substrate surface 110 in a gas phase 200; (3) in the gas phase 200, forming a droplet 30 that adheres to the fiber carrier 10 and includes the hydrogel polymer and the dispersed cells 20 (FIGS. 1B to 1D); The droplets 30 are gelled on the material surface 110 to obtain a fiber carrier-cell-containing gel composite 50 including the fiber carrier 10 and the cell-containing gel body 40 attached to the fiber carrier 10 (see FIG. 1E);

Here, in this specification, the above (2) is attached to the fiber carrier 10 on the base material surface 110 in the gas phase 200 and dispersed with the first hydrogel polymer. (2-2) forming a first droplet 31 containing first cells 21 (FIG. 1B); and (2-2) adhering to the fiber carrier 10 on the substrate surface 110 in the gas phase 200; , forming a second droplet 32 containing a second hydrogel polymer and dispersed second cells 22 and connected to the first droplet 31 (FIG. 1C), and forming a second droplet 32 on the fiber carrier 10. (FIG. 1D), and in (3) above, the droplet connectors 33 are gelled on the substrate surface 110 in the gas phase 200 to form the fibers. The case where a composite 50 including a carrier 10 and a cell-containing gel body 40 attached to the fiber carrier 10 is obtained (FIG. 1E) will be mainly described.

In this method, first, in the above (1), the base material surface 110 on which the fiber carrier 10 is arranged is prepared. The fiber carrier 10 is not particularly limited as long as it has a fibrous shape and is capable of adhering to the droplets 30 and the cell-containing gel body 40. The material constituting the fiber carrier 10 is not particularly limited as long as the effects of the present invention can be obtained, and any material such as organic materials, inorganic materials, metals, ceramics, etc. can be used. As the fiber carrier 10, for example, artificial fibers or natural fibers are preferably used.

Examples of the artificial fibers include one or more selected from the group consisting of artificial organic fibers such as synthetic resin fibers, semi-synthetic fibers, recycled fibers, artificial inorganic fibers such as glass fibers, metal fibers such as stainless steel fibers, and ceramic fibers. is used.

As the natural fiber, for example, one or more fibers selected from the group consisting of animal-derived fibers, plant-derived fibers, and mineral-derived fibers are used. As the animal-derived fiber, for example, human hair or non-human animal hair (including non-human mammal hair, bird hair, insect-derived fibers such as silk fibers) is used. As the plant-derived fiber, for example, cotton fiber or hemp fiber is used. As the fiber carrier 10, biodegradable fibers may be used.

The fiber carrier 10 preferably has flexibility, particularly preferably a flexibility similar to animal hair (in particular human hair). The shape of the fiber carrier 10 is not particularly limited as long as the effects of the present invention can be obtained, but, for example, it is preferable to have a shape similar to animal hair (particularly human hair). The cross-sectional shape of the fiber carrier 10 is not particularly limited as long as the effects of the present invention can be obtained, and may be circular, elliptical, rectangular, or any other arbitrary shape, but is preferably circular or elliptical. . The fiber carrier 10 may be a hollow body (for example, a hollow fiber) or a solid body, but is preferably a solid body.

Each fiber carrier 10 is a single fibrous carrier that exists independently. That is, the fiber carrier 10 is not woven with other fibers, for example. Further, the fiber carrier 10 does not constitute, for example, a sheet-like cloth (for example, a woven fabric or a non-woven fabric).

The cross-sectional area of the fiber carrier 10 (the area of the cross section perpendicular to the longitudinal direction of the fiber carrier 10) is not particularly limited as long as the effects of the present invention can be obtained, but it may be, for example, 100 μm ² or more. , preferably 300 μm ² or more, more preferably 500 μm ² or more, and particularly preferably 1000 μm ² or more. Further, the cross-sectional area of the fiber carrier 10 may be, for example, 1 mm ² or less, preferably 100,000 μm ² or less, more preferably 50,000 μm ² or less, particularly preferably 10,000 μm ² or less. . The cross-sectional area of the fiber carrier 10 may be specified by arbitrarily combining one of the lower limit values mentioned above and one of the upper limit values mentioned above.

The length of the fiber carrier 10 is not particularly limited as long as the effects of the present invention can be obtained, but for example, it may be 5 mm or more, 10 mm or more, or 15 mm or more. The length may be 20 mm or more, 25 mm or more, or 30 mm or more. Further, the length of the fiber carrier 30 may be, for example, 500 mm or less, or 300 mm or less. The length of the fiber carrier 10 may be specified by arbitrarily combining one of the lower limit values mentioned above and one of the upper limit values mentioned above.

The fiber carrier 10 only needs to be arranged on the base material surface 110 so that the droplet 30 can adhere to a part thereof, and the fiber carrier 10 does not need to be in contact with the base material surface 110. Preferably, at least a portion of the fiber carrier 10 is in contact with the base material surface 110.

When at least a part of the fiber carrier 10 is arranged in contact with the base material surface 110, at least a part of the fiber carrier 10 is arranged in removably contact with the base material surface 110. preferable.

The base material 100 is not particularly limited as long as the fiber carrier 10 can be placed on the surface 110 and the droplets 30 and the cell-containing gel body 40 can be formed, and for example, it can be made of resin, glass, ceramic, or metal. It may also be a base material.

It is preferable that at least the portion of the base material surface 110 where the droplets 30 and the cell-containing gel 40 are formed is a water-repellent surface. In this case, the water contact angle of the water-repellent base material surface 110 may be, for example, 90 degrees or more, preferably 100 degrees or more, more preferably 105 degrees or more, and 110 degrees or more. It is particularly preferable that

The water-repellent substrate surface 110 can be formed, for example, by using the substrate 100 of a water-repellent material (e.g., a hydrophobic resin such as a fluorine-containing polymer) and/or by using a water-repellent treatment (e.g., a fluorine-containing functional group, etc.). (modification with hydrophobic functional groups). Moreover, it is preferable that the base material surface 110 is flat.

The gas phase 200 is not particularly limited as long as it is a gas phase, but the gas preferably contains oxygen, and air is preferably used. The gas phase 200 forming the droplet 30 and the gas phase 200 forming the cell-containing gel body 40 may have the same gas composition or may have different gas compositions.

In this method, next, in (2) above, droplets 30 attached to the fiber carrier 10 are formed on the base material surface 110 in the gas phase 200. Specifically, the droplet 30 transfers a suspension of the cells 20 (an aqueous hydrogel solution in which the cells 20 are suspended) containing a hydrogel polymer and dispersed cells 20 to a portion of the fiber carrier 10. It is formed by dropping it onto the base material surface 110 so as to cover it.

More specifically, in the example shown in FIGS. 1B to 1D, in the gas phase 200, on the base material surface 110, as a droplet 30 attached to the fiber carrier 10, a first droplet 31, a second droplet 32, and a droplet connector 33 are formed. The first droplet 31 includes the first cell 21 as the cell 20, the second droplet 32 includes the second cell 22 as the cell 20, and the droplet linkage 33 includes the first cell 21 as the cell 20. It includes a first cell 21 and a second cell 22.

That is, first, a suspension of the first cells 21 containing the first hydrogel polymer is dropped so as to cover a part of the fiber carrier 10, and the first droplets adhering to the fiber carrier 10 are 31, and then a suspension of second cells 22 containing a second hydrogel polymer is applied to cover the other part of the fiber carrier 10 adjacent to the first droplet 32. The liquid is dropped to form a second droplet 32 that adheres to the fiber carrier 10 and is connected to the first droplet 31 . In this way, a droplet link body 33 attached to the fiber carrier 10 is obtained, which is formed by the fusion of the first droplet 31 and the second droplet 32. Dropping of the liquid droplet 30 is preferably performed using a dispensing instrument 300 such as a pipette, as shown in FIGS. 1B and 1C.

A part of the surface of the droplet 30 attached to the fiber carrier 10 is in contact with the base material surface 110. That is, in the example shown in FIGS. 1B to 1D, a portion of the surface of the first droplet 31 is in contact with a portion of the substrate surface 110, and a portion of the surface of the second droplet 32 is in contact with the substrate surface 110. It contacts another part of the material surface 110.

The droplets 30 attached to the fiber carrier 10 have fluidity in the gas phase 200, but contain hydrogel polymers and have higher viscosity and specific gravity than water, so the cells 20 are not dispersed in the droplets 30. maintained in the same state.

That is, in the example shown in FIGS. 1B to 1D, the first cells 21 are maintained in a dispersed state in the portion of the droplet connector 33 originating from the first droplet 31, and the droplet connector 33 is maintained in a dispersed state. In the portion originating from the second droplet 32, the second cells 22 are maintained in a dispersed state.

In the droplet 30, the dispersed individual cells are not bound to other cells or are bound to only a few cells, and are dispersed and suspended in the droplet 30. It is a cell. For example, when a cell suspension containing dispersed cells is centrifuged in order to separate the cells contained in the cell suspension from a solvent, the cell precipitate formed after centrifugation is The cells constituting the precipitate, which are aggregates, are not dispersed cells.

The cell 20 is not particularly limited as long as it is a living cell derived from an animal. The animal may be a human or a non-human animal, but it is preferable to use human cells. The non-human animal is preferably a non-human vertebrate (vertebrate other than human), although it is not particularly limited. The non-human vertebrate is not particularly limited, but is preferably a non-human mammal. 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 mammals. It may also be a hoofed animal (eg, pig, cow, horse, goat, sheep).

The cells 20 may be differentiated cells or undifferentiated cells (stem cells). Stem cells may be totipotent stem cells, pluripotent stem cells, or tissue stem cells. Specifically, the stem cells may be, for example, induced pluripotent (iPS) stem cells, embryonic stem (ES) cells, or embryonic germ (EG) cells. . Differentiated cells are not particularly limited as long as they have differentiated functions, and may be, for example, cells collected from a living body (or cells cultured after being collected from a living body). However, cells derived from stem cells in vitro may also be used.

The cell 20 may be a cell derived from an in-vivo tissue. In vivo tissues include, but are not particularly limited to, hair follicle tissue, skin tissue, liver tissue, heart tissue, kidney tissue, nerve tissue, bone tissue, cartilage tissue, bone marrow tissue, lung tissue, glandular tissue, and periodontal tissue. It may also be tissue or blood.

The cells 20 may be adherent cells or non-adherent cells, but are preferably adherent cells. Adherent cells are cells that exist in a state of adhesion to other cells and/or extracellular matrices in vivo.

As the cell 20, only one type of cell may be used, or two or more types of cells may be used in combination. That is, one droplet 30 may contain only one type of cell, or may contain two or more types of cells. Moreover, one cell-containing gel body 40 may contain only one type of cell, or may contain two or more types of cells.

As shown in FIGS. 1A to 1H, when at least a first cell 21 and a second cell 22 are used, the combination of the first cell 21 and the second cell 22 has the effect of the present invention. Although there is no particular limitation as long as it can be obtained, a combination of different interacting cells is preferred. The combination of different cells is, for example, a combination of cells that differ in one or more selected from the group consisting of differentiation function, proliferative ability, and cell surface marker.

The interaction between the first cell 21 and the second cell 22 is not particularly limited, but may include, for example, a combination in which a substance secreted by one cell acts on the other cell, and/or the formation of an intercellular bond. It is preferable that the combination is Note that when a substance secreted by one cell acts on the other cell, the substance secreted by the other cell may also act on the one cell. Further, when a substance secreted by one cell acts on the other cell, the substance may diffuse inside the cell-containing gel body 40 and act on the other cell, or the substance secreted by the cell may act on the other cell. It may be diffused into a solution (for example, a culture solution) containing the gel body 40, and then act on the other cell of the cell-containing gel body 40 from the solution.

The combination of the first cell 21 and the second cell 22 is preferably a combination of different cells that interact in vivo. In this case, the combination of the first cell 21 and the second cell 22 may be a combination of different cells that interact in the same tissue in the living body. In vivo tissues include, but are not particularly limited to, hair follicle tissue, skin tissue, liver tissue, heart tissue, kidney tissue, nerve tissue, bone tissue, cartilage tissue, bone marrow tissue, lung tissue, glandular tissue, and periodontal tissue. It may also be tissue or blood.

The combination of first cells 21 and second cells 22 may be a combination of cells derived from the same animal or a combination of cells derived from different animals. That is, both the first cell 21 and the second cell 22 may be human cells (cells derived from humans), or both may be non-human animal cells (cells derived from animals other than humans). Alternatively, one may be a human cell and the other may be a non-human animal cell.

The combination of the first cell 21 and the second cell 22 may be, for example, a combination of an epithelial cell and a mesenchymal cell. The combination of epithelial cells and mesenchymal cells is not particularly limited; A combination of acting epithelial cells and mesenchymal cells is preferred.

Epithelial cells and/or mesenchymal cells are cells collected from the hair follicle tissue or glandular tissue (e.g., one or more selected from the group consisting of sweat glands, sebaceous glands, lacrimal glands, and salivary glands) of a living body (e.g., The cells may be cells collected from hair follicle tissue and then cultured, or may be cells derived from stem cells in vitro.

Specifically, the epithelial cells are derived from the outermost layer cells of the outer root sheath 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). The cells may also be hair follicle epithelial cells. Furthermore, the epithelial cells may be epithelial stem cells.

The epithelial cells may have hair growth-related properties (eg, expression of hair growth-related genes and/or formation of hair follicle primordia by co-culture with mesenchymal cells). The epithelial cells may be cells derived from hair follicle tissue (for example, 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), and may be cells derived from skin tissue. Alternatively, the cells may be derived from stem cells (eg, iPS stem cells, ES cells, or EG cells) in vitro.

The epithelial cells may be primary cells collected from a living body, or cells that have been cultured in advance (for example, subcultured cells and/or established cell lines). Epithelial cells are specified, for example, as cells that express cytokeratin. Preferably, the epithelial cells are epithelial stem cells. Epithelial stem cells are identified, for example, as cells expressing cytokeratin 15 and/or CD34.

Mesenchymal cells are dermal papilla cells, dermal root sheath cells, nascent skin mesenchymal cells, or follicular mesenchymal cells derived from stem cells (e.g., iPS cells, ES cells, or EG cells). It may be a thing.

Mesenchymal cells may have hair growth-related properties (eg, expression of hair growth-related genes and/or formation of hair follicle primordia by co-culture with epithelial cells). Mesenchymal cells may be cells derived from adult hair follicle tissue (e.g., dermal papilla and/or hair bulb root sheath), and may be cells derived from skin tissue (fetal, juvenile, or adult skin tissue). The cells may be derived from stem cells (e.g., induced pluripotent (iPS) stem cells, embryonic stem (ES) cells, or embryonic germ (EG) cells) in vitro. The cells may also be

The mesenchymal cells may be primary cells collected from a living body, or cells that have been cultured in advance (for example, subcultured cells and/or established cell lines). Mesenchymal cells are identified, for example, as cells expressing Versican and ALP (alkaline phosphatase). Specifically, as the mesenchymal cells, dermal papilla cells and/or hair bulb sheath cells are preferably used. Dermal papilla cells and bulb sheath cells express Versican and ALP.

In the example shown in FIGS. 1B to 1D, the first droplet 31 mainly contains the first cells 21 as the cells 20, and the second droplet 32 mainly contains the second cells 22 as the cells 20. include. That is, the ratio of the number of first cells 21 to the total number of cells contained in the first droplet 31 and/or the ratio of the number of second cells 22 to the total number of cells contained in the second droplet 32. The proportions may be independently of each other, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, It is particularly preferable that it is 90% or more.

The hydrogel polymer is not particularly limited as long as it is a hydrophilic polymer that has gelling ability. The hydrogel polymer may be a naturally derived polymer or an artificially synthesized polymer, but is preferably a naturally derived polymer. Moreover, it is preferable that the hydrogel polymer is a biocompatible polymer.

Preferably, the hydrogel polymer is an extracellular matrix. The extracellular matrix is not particularly limited as long as it exists in the living body. The hydrogel polymer may have cell adhesive properties to the cells 20.

That is, in the examples shown in FIGS. 1B and 1C, the first hydrogel polymer contained in the first droplet 31 has cell adhesion to the first cells 21 and/or The second hydrogel polymer contained in the second droplet 32 may have cell adhesive properties to the second cells 22.

A hydrogel polymer having cell adhesive properties is a polymer that binds to molecules on the cell surface, such as a specific amino acid sequence that binds specifically or non-specifically to the molecule on the cell surface, and/or It has sugar chains.

Specifically, the hydrogel polymer having cell adhesive properties is, for example, collagen (for example, one or more collagens selected from the group consisting of type I, type II, type III, type IV, type V, and type XI). ), fibronectin, laminin, elastin, glycosaminoglycan (eg, hyaluronic acid), proteoglycan, fibrin, and gelatin.

Further, the hydrogel polymer may be one or more selected from the group consisting of gelatin, agarose, sodium alginate, and synthetic polymers (e.g., polyacrylamide, polyvinyl alcohol, methylcellulose, polyethylene oxide, etc.).

In the example shown in FIGS. 1B and 1C, the first hydrogel polymer contained in the first droplet 31 and the second hydrogel polymer contained in the second droplet are the same type of polymer. It may be a molecule or a different type of polymer. That is, for example, the first hydrogel polymer and the second hydrogel polymer may both be type I collagen, or the first hydrogel polymer may be type I collagen and the second hydrogel polymer may be type I collagen. The hydrogel polymer may be a glycosaminoglycan.

The density of the cells 20 contained in the droplet 30 (in the case of dropping a plurality of droplets 31 and 32, the density of the cells 20 contained in each of the plurality of droplets 31 and 32) is determined by the effect of the present invention. Although it is not particularly limited as long as it is within the range that can be obtained, for example, it may be 1×10 ² cells/mL or more and 1×10 ⁹ cells/mL or less, and 1×10 ³ cells/mL or more and 1× It may be 10 ⁸ cells/mL or less, 1×10 ⁴ cells/mL or more and 1×10 ⁸ cells/mL or less, 1×10 ⁵ cells/mL or more and 1×10 ⁷ It may be less than or equal to cells/mL, or may be greater than or equal to 1×10 ⁶ cells/mL and less than or equal to 1×10 ⁷ cells/mL.

The volume of the droplet 30 (in the case of dropping a plurality of droplets 31, 32, the volume of each of the plurality of droplets 31, 32 (volume of one droplet)) is such that the effect of the present invention can be obtained. There is no particular limitation as long as it is within the range, but for example, it may be 0.01 μL or more and 1 mL or less, 0.1 μL or more and 100 μL or less, or 1 μL or more and 10 μL or less. Good too.

Note that the droplet 30 to be gelled in this method may be a connected droplet 33 formed by fusion of two droplets 31 and 32, as shown in FIGS. 1D and 1E. However, the present invention is not limited to this, and the droplet may be composed of one droplet, or may be a droplet connected body formed by fusing three or more droplets.

In this method, the droplets 30 attached to the fiber carrier 10 are further gelled in the gas phase 200 on the base material surface 110 in (3) above. That is, in the example shown in FIGS. 1D and 1E, the droplet links 33 attached to the fiber carrier 10 are gelled on the base material surface 110 in the gas phase 200.

As a result, a composite 50 including the fiber carrier 10 and the cell-containing gel body 40 attached to the fiber carrier 10 is obtained. That is, in the example shown in FIGS. 1D and 1E, by gelling the droplet connector 33, the first gel part 41 originating from the first droplet 31 and the first gel part 41 originating from the second droplet 32 are formed. A cell-containing gel body 40 including a second gel portion 42 is obtained. In the composite 50, the cell-containing gel body 40, which does not have fluidity, is fixed to the fiber carrier 10.

Like the droplet 30, the cell-containing gel body 40 formed by gelation of the droplet 30 covers a part of the fiber carrier 10, and a part of the surface of the cell-containing gel body 40 is covered with the base material. Contacting surface 110.

Due to gelation, the hydrogel polymer contained in the droplet 30 forms an intermolecular network, the droplet 30 loses fluidity, and a cell-containing gel body 40 is obtained. The gelation method is not particularly limited, and gelation is performed under appropriate conditions depending on the type and/or concentration of the hydrogel polymer contained in the droplet 30, for example. Specifically, for example, when the droplet 30 contains type I collagen as a hydrogel polymer, gelation may be performed by maintaining the droplet 30 at 25° C. to 37° C. for 15 minutes to 60 minutes. Can be done.

The volume of the cell-containing gel body 40 that has not yet been cultured is not particularly limited as long as it is within the range where the effects of the present invention can be obtained, but may be, for example, 0.01 μL or more and 100 mL or less, It may be 1 μL or more and 10 mL or less, 1 μL or more and 1 mL or less, or 1 μL or more and 500 μL or less.

The density of the cells 20 contained in the cell-containing gel body 40 that has not yet been cultured is not particularly limited, but may be, for example, 1×10 ² cells/mL or more and 1×10 ⁹ cells/mL or less. , 1×10 ³ cells/mL or more and 1×10 ⁸ cells/mL or less, 1×10 ⁴ cells/mL or more and 1×10 ⁸ cells/mL or less, 1 It may be 1×10 ⁵ cells/mL or more and 1×10 ⁷ cells/mL or less, or it may be 1×10 ⁶ cells/mL or more and 1×10 ⁷ cells/mL or less.

According to this method, droplets 30 containing hydrogel polymers and dispersed cells 20 attached to fiber carrier 10 are first formed on base material surface 110 in gas phase 200, and then By the simple operation of gelling the droplets 30, the composite 50 containing the cell-containing gel body 40 fixed to the fiber carrier 10 can be easily produced.

Further, as shown in FIGS. 1A to 1E, when connecting a plurality of droplets 31 and 32 on the substrate surface 110 in the gas phase 200, for example, the size of each droplet 31 and 32, and Alternatively, by adjusting the arrangement, the size of the interface for interaction between the plurality of cells 21 and 22 in the cell-containing gel body 40 that is finally formed can be easily and effectively adjusted. can.

The method may further include detaching the cell-containing gel body 40 of the complex 50 from the substrate surface 110. That is, in this case, as shown in FIGS. 1E and 1F, the cell-containing gel body attached to the fiber carrier 10 and in contact with the base material surface 110 is detached from the base material surface 110, and the cell-containing gel body is removed from the base material surface 110. A cell-containing gel body 40 attached to the fiber carrier 10 and not in contact with the surface 110 is obtained.

The method of detaching the cell-containing gel body 40 from the base material surface 110 is a method of detaching the cell-containing gel body 40 from the base material surface 110 while maintaining the survival of the cells 20 contained in the cell-containing gel body 40. For example, a method of immersing the base material surface 110 in contact with the cell-containing gel body 40 in an aqueous solution such as a culture solution, a method of immersing the cell-containing gel body 40 and/or the base material, but are not particularly limited thereto. A method of applying a flow of an aqueous solution such as a culture solution to the surface 110, or a method of lightly touching the cell-containing gel body 40 with an instrument such as a pipette tip in an aqueous solution such as a culture solution is preferably used.

In addition, when the cell-containing gel body 40 detached from the base material surface 110 and collected is used as it is to culture the cells 20 within the cell-containing gel body 40, the cell-containing gel body 40 contains It is preferable to detach the cell-containing gel body 40 from the base material surface 110 without performing an enzyme treatment to decompose the hydrogel polymer.

Note that since the cell-containing gel body 40 of the composite 50 is attached to a part of the fiber carrier 10, when detaching the cell-containing gel body 40 from the base material surface 110, the cell-containing gel body 40 of the fiber carrier 10 must be removed. At least that part will also be removed.

By detaching the cell-containing gel body 40, a composite 50 containing the cell-containing gel body 40 not attached to the base material surface 110 is obtained. The composite 50 including the cell-containing gel 40 that is not attached to anything other than the fiber carrier 10 can be used for various purposes.

The composite body 50 detached from the base material surface 110 has end portions 10a and 10b of the fiber carrier 10 extending from the cell-containing gel body 40, as shown in FIGS. 1F to 1H. The ends 10a, 10b of the fiber carrier 10 extending from the cell-containing gel body 40 in the complex 50 may be both the two ends 10a, 10b of the fiber carrier 10, or the two ends 10a , 10b. Furthermore, the ends 10a and 10b of the fiber carrier 10 extending from the cell-containing gel body 40 of the composite body 50 are free ends, as shown in FIGS. 1F to 1H.

The method may further include culturing the cells 20 within the cell-containing gel body 40 of the complex 50. In this case, the cell-containing gel body 40 of the complex 50 is immersed in a culture solution 410, and the cells 20 are cultured within the cell-containing gel body 40 in the culture solution 410.

Moreover, it is preferable to culture the cells 20 within the cell-containing gel body 40 detached from the base material surface 110. That is, in the example shown in FIGS. 1F to 1H, after the cell-containing gel body 40 is detached from the substrate surface 110, the cell-containing gel body 40 is immersed in the culture solution 410 in the culture container 400, and the cell-containing gel body 40 is immersed in the culture solution 410 in the culture container 400. The first cells 21 and the second cells 22 are cultured within the cell-containing gel body 40 in the liquid 410. More specifically, in the cell-containing gel body 40, the first cells 21 are cultured in the first gel part 41, and the second cells 22 are cultured in the second gel part 42.

Further, as shown in FIGS. 1F to 1H, the entire fiber carrier 10 having free ends (ends 10a, 10b) is immersed in a culture solution 410, and the cell-containing gel body 40 attached to the fiber carrier 10 is The cells 20 may also be cultured.

The culture solution 410 is not particularly limited as long as it is an aqueous solution having characteristics such as composition, pH, and osmotic pressure necessary to maintain the survival of the cells 20. Components contained in the culture solution 410 include, for example, saccharides, amino acids, vitamins, inorganic salts, antibiotics, and growth factors.

The culture time is not particularly limited, and may be, for example, 12 hours or more and 10 days or less, or 1 day or more and 7 days or less. The culture temperature is not particularly limited as long as it can maintain the survival of the cells 20, and may be, for example, 25°C or higher and 40°C or lower, and preferably 30°C or higher and 39°C or lower.

In this method, it is preferable to maintain the cell-containing gel body 40 in a floating state in the culture solution 410 and to culture the cells 20 within the cell-containing gel body 40 in the floating state. That is, in the example shown in FIGS. 1G and 1H, the first cells 21 and the second cells 22 are cultured within the cell-containing gel body 40 floating in the culture solution 410.

Note that the state in which the cell-containing gel body 40 is floating in the culture solution 410 is a state in which the cell-containing gel body 40 is not substantially attached to the wall surface of the culture container 400. That is, for example, if a flow is generated in the culture solution 410, the cell-containing gel body 40 not only floats in the culture solution 410 without contacting the wall surface of the culture container 400, but also easily comes off from the wall surface of the culture container 400. The cell-containing gel body 40, which is weakly attached to the wall surface to the extent that it is separated, is also in a substantially floating state.

In this method, the cells 20 may be cultured within the cell-containing gel body 40 to form aggregates 60 of the cells 20. That is, in this case, by culturing the cells 20 within the cell-containing gel body 40 attached to the fiber carrier 10, cell aggregates 60 attached to the fiber carrier 10 are formed within the cell-containing gel body 40.

That is, when culturing the first cells 21 and second cells 22 within the cell-containing gel body 40, the first cells 21 are aggregated as cell aggregates 60 within the cell-containing gel body 40. A lump 61 and/or an aggregate 62 of the second cells 22 is formed.

Specifically, in the example shown in FIGS. 1G and 1H, the first gel part 41 is cultured in the cell-containing gel body 40 attached to the fiber carrier 10. At the same time, an aggregate 61 of the first cells 21 attached to the fiber carrier 10 is formed, and an aggregate 62 of the second cells 22 attached to the fiber carrier 10 is formed at the second gel part 42. ing.

The cell aggregate 60 is formed by the cells 20 bonding to each other and spontaneously aggregating within the cell-containing gel body 40 over the course of culture time. That is, in the example shown in FIGS. 1G and 1H, the first cells 21 aggregate to form the first cell aggregate 61 in the cell-containing gel body 40, and the second cells 22 aggregate to form the first cell aggregate 61. A second cell aggregate 62 is formed.

Alternatively, as shown in FIGS. 1G and 1H, a first cell aggregate 61 and a second cell aggregate 62 may be formed which are bonded to each other. In this case, some of the first cells 21 forming the first cell aggregate 61 and some of the second cells 22 forming the second cell aggregate 62 A second cell 22 forms a bond. As a result, within the cell-containing gel body 40, a connected cell aggregate 63 that includes the first cell aggregate 61 and the second cell aggregate 62 and is attached to the fiber carrier 10 is formed. In this cell aggregate connected body 63, the first cell aggregate 61 and the second cell aggregate 62 are arranged in the direction in which the fiber carrier 10 extends (the longitudinal direction of the fiber carrier 10), as shown in FIG. 1H. connected.

Specifically, for example, the first cell 21 is an epithelial cell (for example, a hair follicle epithelial cell such as an epithelial stem cell derived from hair follicle tissue), and the second cell 22 is a mesenchymal cell (for example, In the case of hair follicle mesenchymal cells such as dermal papilla cells, the first cells 21 form a first cell aggregate 61 within the cell-containing gel body 40 attached to the fiber carrier 10, and the first cells 21 form a first cell aggregate 61. The second cells 22 form a second cell aggregate 62, and the first cell aggregate 61 and the second cell aggregate 62 are connected to each other, thereby forming cells that are hair follicle primordia. A connected aggregate 63 is formed. The connected cell aggregate 63, which is the hair follicle primordium, preferably has the ability to grow hair when transplanted into a living body.

In this method, the cell-containing gel body 40 containing the dispersed cells 20 may be cultured to obtain the contracted cell-containing gel body 40 containing the aggregates 60 of the cells 20. That is, when the cells 20 in the cell-containing gel body 40 bond to each other and gradually aggregate to form a cell aggregate 60 as the culture time passes, the cell-containing gel body 40 Accordingly, it contracts while remaining attached to the fiber carrier 10.

Specifically, as shown in FIGS. 1G and 1H, by culturing the cell-containing gel body 40 containing the dispersed first cells 21 and the dispersed second cells 22, the first cells 21 A shrunken cell-containing gel body 40 containing an aggregate 61 of the second cells 22 and an aggregate 62 of the second cells 22 is obtained.

Cell-containing gel body 40 containing dispersed cells 20 at the start of culture (for example, cell-containing gel body 40 containing dispersed first cells 21 and dispersed second cells 22 shown in FIG. 1G). Volume of the cell-containing gel body 40 containing the cell aggregate 60 after culture (for example, the cell-containing gel body 40 containing the first cell aggregate 61 and the second cell aggregate 62 shown in FIG. 1H) with respect to the volume For example, the ratio may be 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. It may be a thing.

Such shrinkage of the cell-containing gel body 40 as the culture progresses becomes significant when the hydrogel polymer has cell adhesive properties to the cells 20. That is, the contraction of the cell-containing gel body 40 as shown in FIGS. 1G and 1H occurs because the first hydrogel polymer has cell adhesive properties to the first cells 21 and/or This becomes noticeable when the second hydrogel polymer has cell adhesion to the second cells 22.

The cell aggregate 60 is produced by detaching the cell-containing gel body 40 from the base material surface 110 and then culturing the cells 20 within the cell-containing gel body 40 floating in the culture solution 410. is formed.

The density of the cells 20 in the cell-containing gel body 40 containing the cell aggregates 60 (the number of cells 20 contained in a unit volume of the cell-containing gel body 40) is, for example, 1×10 ² cells/mL or more, 1×10 ¹¹ cells/mL or less, 1×10 ³ cells/mL or more and 1×10 ¹⁰ cells/mL or less, 1×10 ⁴ cells/mL or more, 1×10 ¹⁰ cells /mL or less, and may be 1 x 10 ⁵ cells/mL or more and 1 x 10 ⁹ cells/mL or less, and 1 x 10 ⁶ cells/mL or more and 1 x 10 ⁹ cells/mL. It may be the following.

In this method, cells 20 are cultured in a cell-containing gel body 40 attached to a fiber carrier 10, and a cell aggregate 60 and a hydrogel covering part 40a covering the cell aggregate 60 are formed. It is also possible to obtain a cell-containing gel body 40 in which the density of the hydrogel polymer inside the mass 60 is higher than that in the hydrogel covering portion 40a.

That is, in the example shown in FIG. 1H, the first cells 21 and the second cells 22 are cultured in the cell-containing gel body 40, and the following characteristics (a) and/or (b) are obtained: (a) first a cell aggregate 61 and a first hydrogel coating portion 41a that covers the first cell aggregate 61, and the density of the first hydrogel polymer inside the first cell aggregate 61 is , larger than that in the first hydrogel coating part 41a; (b) including a second cell aggregate 62 and a second hydrogel coating part 42a covering the second cell aggregate 62; A cell-containing gel body 40 is obtained in which the density of the second hydrogel polymer inside the second cell aggregate 62 is higher than that in the second hydrogel coating portion 42a.

That is, when the cells 20 aggregate to form a cell aggregate 60 within the cell-containing gel body 40 attached to the fiber carrier 10 as the culture time progresses, the hydrogel portion between the aggregated cells 20 is It shrinks significantly compared to the hydrogel portion covering the cell aggregate 60.

As a result, in the cell-containing gel body 40 including the cell aggregates 60, the density of the hydrogel polymer inside the cell aggregates 60 is greater than that in the hydrogel coating portion 40a.

Such local concentration of the hydrogel polymer becomes particularly noticeable when the hydrogel polymer has cell adhesion to the cells 20. That is, in this case, the cells 20 aggregate while pulling together the hydrogel polymers to which they are attached, so that the hydrogel polymers near the cells 20 are attached to the hydrogel polymers distant from the cells 20. It is significantly concentrated compared to

The density distribution of the hydrogel polymer within the cell-containing gel body 40 can be confirmed, for example, by using a staining method specific to the hydrogel polymer. Specifically, for example, the hydrogel polymer contained in the cell-containing gel body 40 is stained with a fluorescently labeled antibody, and the stained cell-containing gel body 40 is observed under a fluorescence microscope to determine cell aggregation. By comparing the fluorescence intensity inside the mass 60 and the fluorescence intensity at the hydrogel coating part 40a, it is possible to determine the density of the hydrogel polymer inside the cell aggregate 60 and the hydrogel polymer in the hydrogel coating part 40a. can be compared quantitatively with the density of

Specifically, in the cell-containing gel body 40 including the cell aggregate 60, the density of the hydrogel polymer inside the cell aggregate 60 relative to the density of the hydrogel polymer in the hydrogel coating portion 40a is, for example, It may be twice or more, five times or more, or ten times or more.

Such uneven distribution of the hydrogel polymer in the cell-containing gel body 40 is achieved by first forming the cell-containing gel body 40 containing dispersed cells 20 in the gas phase 200, and then forming the cell-containing gel body 40 in the gas phase 200. This is due to the operation characteristic of this method, in which the cells 20 are cultured in a cell to form a cell aggregate 60.

In this method, first, a plurality of droplets 31 and 32 are connected and gelled in a gas phase 200 to form a cell-containing gel body 40, and then cells 21 and 22 are cultured within the cell-containing gel body 40. When forming a plurality of cell aggregates 61, 62 bonded to each other, for example, by adjusting the size and/or arrangement of the plurality of droplets 31, 32, the cell-containing gel finally obtained can be adjusted. The size of the bonding surface between the plurality of cell aggregates 61 and 62 in the body 40 can be easily and effectively controlled.

In addition, in the cell-containing gel body 40 obtained as described above, there is an interface between the first cells 21 and the second cells 22 and/or the first cell aggregate 61. Size of the interface for interaction with the second cell aggregate 62 (if the first cell aggregate 61 and the second cell aggregate 62 are bonded, the bonding surface) can be set arbitrarily.

FIGS. 2A to 2H schematically show, in plan view, the main steps included in another example of the present method. 3A to 3H schematically show cross sections taken along lines III-A to III-H shown in FIGS. 2A to 2H.

In the above method (1), a plurality of fiber carriers 10 arranged on the base material surface 110 are prepared, and the above (2) and (3) are carried out for each of the plurality of fiber carriers 10. You can also use it as

That is, in the examples shown in FIGS. 2A to 2E and 3A to 3E, first, a plurality (three) of fiber carriers 10 arranged on the base material surface 110 are prepared, and then the plurality (three) of fiber carriers 10 are prepared. For each of the fiber carriers 10, formation of a droplet 30 attached to the fiber carrier 10 and formation of a cell-containing gel body 40 by gelation of the droplet 30 are performed, so that each fiber carrier 10 A plurality of (three) composites 50 including cell-containing gel bodies 40 attached to the cell-containing gel body 40 are manufactured.

Note that the plurality of fiber carriers 10 are preferably arranged in parallel on the base material surface 110. Further, even when a plurality of fiber carriers 10 are arranged on the base material surface 110, each cell-containing gel body 40 is formed so as to adhere to only one of the plurality of fiber carriers 10. That is, the number of fiber carriers 10 to which each cell-containing gel body 40 is attached is one. Thus, each composite 50 produced by the method includes one fiber carrier 10. That is, the number of fiber carriers 10 included in each composite 50 is one.

In the method (2) above, in the gas phase 200, on the substrate surface 110, each fiber carrier 10, each containing a hydrogel polymer and dispersed cells 20, is attached to one fiber carrier 10 at a distance from each other. A plurality of droplets 30 are formed, and in (3) above, the droplets 30 are gelled on the base material surface 110 in the gas phase 200, and the droplets 30 are gelled on the one fiber carrier 10 and separated from each other. A composite 50 including a plurality of cell-containing gel bodies 40 attached to one fiber carrier 10 may be obtained.

That is, in the examples shown in FIGS. 2C to 2E and 3C to 3E, each fiber carrier 10 was first attached to one fiber carrier 10 at a distance from each other on the base material surface 110 in the gas phase 200. A plurality of (three) droplets 30 are formed, and then the droplets 30 are gelled to form a plurality of (three) droplets attached to the one fiber carrier 10 and spaced apart from each other. ) and a cell-containing gel body 40 is manufactured.

More specifically, regarding each of the plurality of (three) fiber carriers 10 arranged on the base material surface 110, in the above (2-1), each of the fiber carriers 10 is A plurality (three) of first droplets 31 containing a first hydrogel polymer and dispersed first cells 21 are attached to one fiber carrier 10 at a distance from each other (Fig. 2C and FIG. 3C), in (2-2) above, in the gas phase 200, on the substrate surface 110, each containing the second hydrogel polymer and the second cells 22 dispersed therein, and A plurality of (three) second droplets 32 connected to different ones of the plurality of (three) first droplets 31 and attached to the one fiber carrier 10 spaced apart from each other are formed to A plurality (three) of connected droplets 33 are obtained which are spaced apart and adhered to the one fiber carrier 10 (FIGS. 2D and 3D), and in (3) above, on the base material surface 110 in the gas phase 200. , the plurality (three) of the droplet connected bodies 33 are gelled to form the one fiber carrier 10 and the plurality (three) cell-containing gel bodies that are spaced apart from each other and adhered to the one fiber carrier 10. 40 is obtained (FIGS. 2E and 3E).

Note that for dropping the plurality of droplets 30 attached to the fiber carrier 10, an automatic dispensing robot equipped with a plurality of regularly arranged dispensing instruments (for example, a plurality of pipette tips) is preferably used. Such an automatic dispensing robot has, for example, a tank containing a suspension containing a hydrogel polymer and dispersed cells 20, and a stage on which the base material 100 is placed, and its operation is controlled by a computer. controlled.

Then, by operating the automatic dispensing robot, the cell suspension in the tank is transferred from a plurality of regularly arranged dispensing instruments onto the fiber carrier 10 disposed on the surface 110 of the base material 100. By dropping the droplets at a plurality of positions spaced apart from each other, it is possible to efficiently form a plurality of droplets 30 spaced apart from each other and attached to the fiber carrier 10 in a regular arrangement corresponding to the arrangement of the dispensing device. can.

In the method (1) above, a fiber carrier 10 is prepared in which a part is fixed to the support member 500 and the other part is arranged on the base material surface 110, and the fiber carrier 10 is fixed to the support member 500. Regarding the fiber carrier 10, the above (2) and (3) may be implemented.

In this case, the part of the fiber carrier 10 fixed to the support member 500 is not particularly limited as long as it is a part of the fiber carrier 10, but one end 10a and/or the other end of the fiber carrier 10 is not particularly limited. Part 10b is preferred, and both ends 10a and 10b of the fiber carrier 10 are particularly preferred.

In addition, the other part arranged on the base material surface 110 of the fiber carrier 10 fixed to the support member 500 is especially a part other than the part of the fiber carrier 10 fixed to the support member 500. Although not limited to this, when both ends 10a and 10b of the fiber carrier 10 are fixed to the support member 500, the central part 10c, which is the part between the ends 10a and 10b, is fixed to the base material surface 110. Place it on top. At least a portion (for example, the portion to which the droplet 30 adheres) of the portion (for example, the central portion 10c) of the fiber carrier 10 disposed on the base material surface 110 may be disposed in contact with the base material surface 110. preferable.

Alternatively, a plurality of fiber carriers 10 may be provided, each of which has a portion fixed to the support member 500 and another portion of which is disposed on the base material surface 110. In this case, the plurality of fiber carriers 10 are preferably fixed to the support member 500 so as to extend in parallel.

Specifically, in the example shown in FIGS. 2A to 2H, a plurality of (three) fibers each having both end portions 10a and 10b fixed to a support member 500 and a central portion 10c disposed on the base material surface 110 are used. A carrier 10 is used. Further, a plurality of (three) fiber carriers 10 are fixed to the support member 500 in parallel with each other. Note that the plurality of fiber carriers 10 are fixed to the support member 500 at a distance from each other.

In this method, a fiber carrier-cell structure including a fiber carrier 10 to which a cell-containing gel body 40 containing a hydrogel polymer and cells 20 is attached, and a support member 500 to which the fiber carrier 10 is fixed. A kit for producing a containing gel complex (hereinafter referred to as "this kit") is preferably used.

As shown in FIGS. 2A and 3A, this kit includes a plurality of fiber carriers 10 to which cell-containing gel bodies 40 containing hydrogel polymers and cells 20 are attached, and a plurality of fiber carriers 10 separated from each other. The support member 500 may be fixed to the support member 500.

In this method, first, a support member 500 to which a plurality of fiber carriers 10 are fixed is prepared. In the example shown in FIGS. 2A and 3A, the support member 500 is formed between a pair of fixing parts 510a, 510b that fixes the pair of ends 10a, 10b of the fiber carrier 10, and the pair of fixing parts 510a, 510b. and an opening 520. In this example, the opening 520 is formed as a through hole, but may be formed as a hole with a bottom. Further, in the example shown in FIGS. 2A and 3A, the pair of fixing parts 510a and 510b of the support member 500 are connected by a connecting part 530. That is, the support member 500 is formed into a frame shape.

Next, as shown in FIGS. 2B and 3B, the fiber carrier 10 fixed to the support member 500 is placed on the surface 110 of the base material 100. That is, the base material 100 formed separately from the support member 500 is used in combination with the support member 500.

Specifically, in the example shown in FIGS. 2B and 3B, the fiber carrier 10 (specifically Specifically, the central portion 10c) of the fiber carrier 10 is placed on the surface 110 of the substrate 100.

That is, by arranging the support member 500 and the base material 100 in the opening 520 of the support member 500, the fiber carrier 10 fixed to the support member 500 can be placed on the surface 110 of the base material 100. It is formed in such a size and shape that it can be placed in the

In this regard, in this kit used in the present method, in addition to the fiber carrier 10 and the support member 500, the fiber carrier 10 fixed to the support member 500 is placed on the surface 110 of the gas phase 200. In addition, the fiber carrier 10 may further include a base material 100 on which the cell-containing gel body 40 attached to the fiber carrier 10 is formed.

In this case, in addition to the plurality of fiber carriers 10 and the support member 500 to which the plurality of fiber carriers 10 are fixed at a distance from each other, the present kit includes, on the surface 110, each of the plurality of fiber carriers 10. It may further include a base material 100 on which a plurality of cell-containing gel bodies 40 are formed.

In the present method, in (2) above, the cells 20 attached to the fiber carrier 10 fixed to the support member 500 and dispersed in the hydrogel polymer are formed on the base material surface 110 in the gas phase 200. In (3) above, the droplets 30 are gelled on the base material surface 110 in the gas phase 200, and the fiber carrier 10 fixed to the support member 500 and the target A fiber carrier-cell-containing gel composite 50 including a cell-containing gel body 40 attached to a fiber carrier 10 is obtained.

In this method, when using a support member 500 to which a fiber carrier 10 is fixed, the cell-containing gel body 40 attached to the fiber carrier 10 fixed to the support member 500 is detached from the base material surface 110. It may also be included.

That is, as shown in FIGS. 2D to 2E and 3D to 3E, a cell-containing gel body 40 is formed on the base material surface 110 in the gas phase 200, adhering to the fiber carrier 10 fixed to the support member 500. After obtaining the composite 50, the cell-containing gel body 40 attached to the fiber carrier 10 is detached from the base material surface 110 while the fiber carrier 10 is fixed to the support member 500. In this case, the composite 50 is a cell-containing gel body attached to the fiber carrier 10 fixed to the support member 500 and detached from the base material surface 110 (for example, floating in the gas phase or liquid phase). This includes 40.

In this method, the cells 20 may be cultured within the cell-containing gel body 40 attached to the fiber carrier 10 fixed to the support member 500.

That is, as shown in FIGS. 2F to 2G and 3F to 3G, cells 20 are cultured within a plurality of cell-containing gel bodies 40 attached to each of a plurality of fiber carriers 10 fixed to a support member 500.

Specifically, in the examples shown in FIGS. 2F to 2G and 3F to 3G, the cell-containing gel body 40 attached to each fiber carrier 10 fixed to the support member 500 is placed in the culture medium 410 in the culture container 400. The cells 20 are cultured in the cell-containing gel body 40 that is immersed and floats in the culture solution 410 .

More specifically, in the examples shown in FIGS. 2F to 2G and 3F to 3G, at least the cell-containing gel body 40 and the fiber carrier 10 fixed to the support member 500 are attached. 410 is immersed in the culture solution 410. In this example, the entire composite 50 fixed to the support member 500 is immersed in the culture solution 410. Furthermore, the fixing parts 510a and 510b of the support member 500 to which the fiber carrier 10 is fixed are also immersed in the culture solution 410. However, the present invention is not limited to these examples, as long as the cell-containing gel body 40 attached to the fiber carrier 10 fixed to the support member 500 is placed in the culture solution.

In this method, the cells 20 may be cultured within the cell-containing gel body 40 attached to the fiber carrier 10 fixed to the support member 500 to form the cell aggregate 60. That is, in the examples shown in FIGS. 2F to 2G and 3F to 3G, the first cells 21 and the second cells 22 are placed in the cell-containing gel body 40 attached to the fiber carrier 10 fixed to the support member 500. The cells are cultured to form aggregates 61 of the first cells 21 and aggregates 62 of the second cells 22.

When forming cell aggregates 60 attached to the fiber carrier 10 fixed to the support member 500, as shown in FIGS. 2F to 2G and 3F to 3G, the cells at the start of culture, including the dispersed cells 20, Compared to the cell-containing gel body 40 (FIGS. 2F and 3F), the cell-containing gel body 40 (FIGS. 2G and 3G) containing the cell aggregates 60 after culture shrinks and decreases in volume. good.

In this method, when forming a composite 50 including a plurality of cell-containing gel bodies 40 attached to one fiber carrier 10, this method involves cutting the one fiber carrier 10 of the composite 50, One fiber carrier piece 11 (a part of the fiber carrier 10 having free ends 11a and 11b) and one cell-containing gel body 40 attached to the one fiber carrier piece 11 (a part of the fiber carrier 10 having free ends 11a and 11b). Obtaining a fiber carrier-cell-containing gel composite piece 51 (a part of the composite 50 including the plurality of cell-containing gel bodies 40) containing one of the plurality of cell-containing gel bodies 40 attached thereto. It is good also as including further.

Specifically, in the example shown in FIGS. 2H and 3H, the portion between the plurality of cell-containing gel bodies 40 spaced apart from each other in one fiber carrier 10 fixed to the support member 500 included in the composite 50 is cut to obtain a composite piece 51.

More specifically, of one fiber carrier 10 fixed to the support member 500, the part between adjacent cell-containing gel bodies 40 attached to the one fiber carrier 10 (the part between the adjacent cell-containing gel bodies 40) By cutting the connecting portion), a composite piece 51 including one of the adjacent cell-containing gel bodies 40 and one fiber carrier piece 11 that is a part of the fiber carrier 10 is obtained. Note that both ends 11a and 11b of the fiber carrier piece 11 included in the composite piece 51 isolated by cutting become free ends.

In the examples shown in FIGS. 2H and 3H, one composite piece 51 is formed from each of the three composites 50, but each of the three composites 50 is made of cell-containing gel. Since each composite body 40 includes three bodies 40, a maximum of nine composite pieces 51 can be obtained by further cutting the fiber carrier 10 fixed to the support member 500. That is, for some or all of the plurality of composites 50, the fiber carrier 10 to which a predetermined number of cell-containing gel bodies 40 attached, which are included in one composite 50, is cut is cut, and the predetermined number of composite pieces 51 are cut. It is also possible to obtain .

Note that when the fiber carrier 10 is fixed to the support member 500, the fiber carrier 10 fixed to the support member 500 is cut, but the present invention is not limited to this. For example, when the support member 500 is not used, multiple fiber carriers 10 are cut. The fiber carrier 10 having both free end portions 10a and 10b to which the cell-containing gel body 40 is attached may be cut.

As shown in FIGS. 2H and 3H, the timing of cutting one fiber carrier 10 to which a plurality of cell-containing gel bodies 40 are attached is determined after the cells 20 are cultured in the cell-containing gel body 40, especially when the cells aggregate. Although it is preferable to do this after the mass 60 is formed, the timing is not limited to this, and for example, it may be performed at any timing during culturing of the cells 20 in the cell-containing gel body 40, or at any timing after the cell-containing gel body 40 is cultured. It may be before the start of culturing the cells 20 in the cell 40.

According to this method, a composite 50 including a fiber carrier 10 and a cell-containing gel body 40 attached to the fiber carrier 10 is easily produced. The composite 50 produced by this method preferably includes one fiber carrier 10 and one cell-containing gel body 40 containing a hydrogel polymer and cells 20 and attached to the one fiber carrier.

The composite 50 containing only one cell-containing gel body 40 may be produced by attaching only one droplet 30 or droplet link 33 to one fiber carrier 10 and gelling it, as described above. Alternatively, the composite piece 51 may be formed by cutting one fiber carrier 10 to which a plurality of cell-containing gel bodies 40 are attached at a distance from each other. That is, when cutting one fiber carrier 10 to which a plurality of cell-containing gel bodies 40 are attached, this method may be a method for manufacturing composite pieces 51 as composites 50.

In the complex 50, each cell-containing gel body 40 may include cell aggregates 60, as described above. The composite 50 produced by this method comprises one fiber carrier 10 and a plurality of cell-containing gel bodies 40, each containing a hydrogel polymer and cells 20, attached to the one fiber carrier at a distance from each other. , may also be included.

Furthermore, as described above, in the complex 50, the cell-containing gel body 40 includes the first gel part 41 containing the first hydrogel polymer and the first cells 21, and the first gel part 41 containing the first hydrogel polymer and the first cells 21; A second gel portion 42 containing two cells 22 may also be included.

In this case, the first gel part 41 may include the first cell aggregate 61, and the second gel part 42 may include the second cell aggregate 62. Furthermore, the cell-containing gel body 40 may include a connected cell aggregate 63 formed by connecting a first cell aggregate 61 and a second cell aggregate 62.

In the complex 50, the combination of the first cell 21 and the second cell 22 may be a combination of an epithelial cell and a mesenchymal cell. In this case, the cell-containing gel body 40 contained in the complex 50 preferably includes a first cell aggregate 61 and a second cell aggregate 62, and the first cell aggregate 61 and the second cell aggregate It is preferable to include a connected cell aggregate 63 formed by connecting with a cell aggregate 62, and it is particularly preferable to include the connected cell aggregate 63 that has the ability to grow hair when transplanted into a living body.

One fiber carrier 10 included in the composite 50 may be fixed to the support member 500, and in this case, it is preferable that both ends 10a, 10b of the fiber carrier 10 are fixed to the support member 500.

The composite 50 produced by this method can be applied to various uses by taking advantage of the feature that it includes the fiber carrier 10. That is, for example, when the cell-containing gel body 40 of the complex 50 contains cells for transplantation into a living body, the complex 50 is used as a graft. That is, this method may be a method for manufacturing the transplantation composite 50. The living body to which the composite 50 is transplanted may be a human or a non-human animal, but is preferably a human.

In the complex 50, the fiber carrier 10 is effectively utilized as a carrier for transporting the cell-containing gel body 40. That is, for example, when transporting a microscopic cell-containing gel body 40 to a transplant site in a living body, the user grasps the fiber carrier 10 attached to the cell-containing gel body 40 with an instrument such as tweezers, thereby removing the cells. The containing gel body 40 can be transported easily and reliably.

When the cell-containing gel body 40 of the complex 50 contains cells derived from hair follicle tissue or cells derived from glandular tissue (for example, epithelial cells and/or mesenchymal cells), the complex 50 contains hair follicle tissue-derived cells or glandular tissue-derived cells (for example, epithelial cells and/or mesenchymal cells). Alternatively, it can be transplanted into a living body to regenerate glandular tissue. In this case, the fiber carrier 10 is expected to serve as a carrier for the cells 20 contained in the cell-containing gel body 40 attached to the fiber carrier 10 to proliferate and/or migrate.

When the complex 50 produced by the present method is transplanted into a living body and hair grows from the transplanted site (for example, the cell-containing gel body 40 of the complex 50 contains epithelial cells and/or mesenchymal cells, preferably contains an epithelial cell aggregate and/or a mesenchymal cell aggregate, particularly preferably a hair follicle primordium formed by connecting an epithelial cell aggregate and a mesenchymal cell aggregate ), the living organism is not particularly limited and may be a human or a non-human animal, but is preferably a human. In this case, the composite 50 is a hair regeneration graft that has the ability to grow hair when transplanted into a living body. The composite 50 is preferably transplanted to the skin of the living body. Transplantation to the skin may be, for example, subcutaneous or intradermal transplantation. That is, composite 50 may be a skin (eg, scalp) implant.

The production of the composite 50 and its implantation into a living body may be for medical use or for research use. That is, for example, if the complex 50 is manufactured for the purpose of being transplanted into a human patient suffering from or likely to suffer from the disease for the treatment or prevention of a disease accompanied by hair loss, the complex 50 is implanted into the human patient. It may also be possible to port it to

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

Further, for example, the complex 50 is manufactured or the complex 50 is used to search for a substance that can be used for the treatment or prevention of a disease accompanied by hair loss, and/or to search for a substance involved in the mechanism of the disease. It may also be transplanted into a non-human animal.

Note that after the composite 50 is transplanted into a living body, the fiber carrier 10 included in the composite 50 is removed. That is, for example, by pulling out the fiber carrier 10 extending from the implanted composite body 50 using a tool such as tweezers, the fiber carrier 10 can be removed. Further, after the composite 50 is implanted, the fiber carrier 10 of the composite 50 may fall off naturally. Further, when the fiber carrier 10 is made of a biodegradable material, the fiber carrier 10 is decomposed by hydrolysis or the like and removed after implantation.

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

[Preparation of cells]
Dorsal skin tissue was collected from an 18-day-old C57BL/6 mouse fetus and treated with dispase using the method reported by Nakao et al. (Koh-ei Toyoshima et al. Nature Communication, 3, 784, 2012) with some modifications. The skin tissue was shaken at 30 rpm for 1 hour at 4° C. to separate the epithelial layer and mesenchymal layer of the skin tissue. Thereafter, the epithelial layer was treated with collagenase at 100 U/mL for 1 hour and 20 minutes, and then treated with trypsin for 10 minutes to obtain dispersed epithelial cells that had finally passed through a 40 μm mesh cell strainer. Further, the mesenchymal layer was treated with 100 U/mL collagenase for 1 hour and 20 minutes, and finally, dispersed mesenchymal cells that passed through a 40 μm mesh cell strainer were obtained.

[Manufacture of composite]
The water-repellent surface inside the lid of a culture dish with a diameter of 6 cm was used as the substrate surface. As the fiber carrier, a medical suture thread (diameter of about 50 μm, made of nylon or silk) cut into lengths of about 5 cm to 10 cm was used. Then, a plurality of fiber carriers were arranged in parallel on the surface of the base material.

Collagen (Collagen Type I-A, manufactured by Nitta Gelatin Co., Ltd.) was used as the first hydrogel polymer and the second hydrogel polymer. Furthermore, mesenchymal cells were used as the first cells, and epithelial cells were used as the second cells.

That is, mesenchymal cells were dispersed in an aqueous collagen solution at a concentration of 1×10 ⁴ cells/2 μL to prepare a suspension of the mesenchymal cells containing collagen. Similarly, epithelial cells were dispersed in an aqueous collagen solution at a concentration of 1×10 ⁴ cells/2 μL to prepare a suspension of the epithelial cells containing collagen.

First, 2 μL of a suspension of mesenchymal cells was dropped onto each fiber carrier placed on the surface of the base material in the air, and each fiber carrier contained collagen and dispersed mesenchymal cells. A plurality of first droplets were formed that adhered to the fiber carrier.

Next, 2 μL of the epithelial cell suspension was dropped in the air at a position connected to the first droplet on each fiber carrier disposed on the surface of the base material, and each layer was filled with collagen and dispersed epithelium. A plurality of second droplets were formed containing lineage cells, connected to one first droplet, and attached to one fiber carrier.

In this way, a plurality of connected droplets each formed by connecting one first droplet and one second droplet on one fiber carrier and attached to the one fiber carrier are formed. Obtained.

Furthermore, the connected droplets attached to each fiber carrier on the surface of the base material were incubated at 37° C. for 30 minutes to gel them. In this way, a plurality of composites were obtained, each comprising one fiber carrier and one cell-containing gel body attached to the one fiber carrier.

Thereafter, the complex (fiber carrier and cell-containing gel body) is detached from the substrate surface 110 by immersing the complex on the substrate surface in a culture solution and flowing the culture solution by pipetting. I let it happen.

The detached complex was suspended in a culture medium (mixed medium (DMEM (Sigma) + KG2 + 10% fetal bovine serum + 1% penicillin)), and mesenchymal cells and epithelial cells were incubated in the cell-containing gel body of the complex. Cultured for 1 day.

[result]
FIGS. 4A and 4B show phase contrast micrographs of the complex on the first day of culture (Day 1) and the third day of culture (Day 3), respectively. FIGS. 5A and 5B show fluorescence micrographs of the complex on the first day of culture (Day 1) and the third day of culture (Day 3), respectively.

Note that FIGS. 5A and 5B correspond to FIGS. 4A and 4B, respectively. In addition, in Figures 5A and 5B, mesenchymal cells are stained with the first fluorescent reagent (Vybrant (trademark) DiI Cell-Labeling Solution, Invitrogen (trademark)) and detected as red fluorescence, and epithelial cells are detected as red fluorescence. It was stained with a second fluorescent reagent (Vybrant™ DiO Cell-Labeling Solution, Invitrogen™) and detected as green fluorescence.

As shown in FIGS. 4A and 5A, the complex on the first day of culture includes one fiber carrier and a cell-containing gel body, and the cell-containing gel body is dispersed with dispersed mesenchymal cells. It contained epithelial cells.

On the other hand, as shown in FIGS. 4B and 5B, the cell-containing gel body attached to one fiber carrier of the complex on the third day of culture consists of an aggregate of mesenchymal cells and an aggregate of epithelial cells connected to each other. It included.

In addition, the aggregates of mesenchymal cells and epithelial cells covered part of the outer periphery of the fiber carrier. That is, in the composite, the fiber carrier extended to penetrate the mesenchymal cell aggregate and the epithelial cell aggregate.

Furthermore, after 3 days of culture, the cell-containing gel body attached to the fiber carrier contracted significantly. That is, the volume of the cell-containing gel body on the third day of culture was significantly decreased compared to the volume of the cell-containing gel body at the start of culture and on the first day of culture.

[Preparation of cells]
Mesenchymal cells and epithelial cells were prepared in the same manner as in Example 1 above.

[Manufacture of composite]
In the same manner as in Example 1 above, one fiber carrier consisting of a medical suture thread (about 50 μm in diameter, made of nylon, manufactured by Natsume Seisakusho Co., Ltd.) with a length of about 5 mm to 10 mm was attached to the one fiber carrier. A composite containing one cell-containing gel body was produced. The cell-containing gel body of the complex included a connected cell aggregate formed by connecting an aggregate of mesenchymal cells and an aggregate of epithelial cells.

[Transplantation of complex]
The composites prepared as described above were implanted subcutaneously into nude mice. That is, first, in each of a plurality of complexes, one free end of a fiber carrier extending from an aggregate of mesenchymal cells included in a connected cell aggregate, and an epithelium included in the connected cell aggregate. Among the other free ends of the fiber carrier extending from the aggregate of mesenchymal cells, one free end extending from the aggregate of mesenchymal cells was cut.

On the other hand, nude mice were anesthetized by isoflurane inhalation, and their backs were disinfected with isodine. Subsequently, a plurality of graft wounds were formed from the epidermal layer to the lower dermal layer of the skin of the nude mouse using a V-Lance microsurgical knife (manufactured by Nippon Alcon Co., Ltd.).

Then, the free end of the fiber carrier extending from the aggregate of epithelial cells of each complex is grasped with tweezers, and one complex containing the cell-containing gel attached to the fiber carrier is placed in each of the plurality of transplant wounds. injected one by one. That is, in each transplant site, an aggregate of mesenchymal cells is placed inside the skin, an aggregate of epithelial cells is placed outside the skin, and a fiber carrier extending from the aggregate of epithelial cells is placed inside the skin. The composite was implanted so that the free end extended outside the skin. After implantation of the composite, the implanted regions were covered with surgical tape in order to prevent the fiber carriers extending from each implanted region from detaching. Furthermore, animal breeding and animal experiments were conducted in compliance with the guidelines of the Yokohama National University Animal Experiment Committee.

In addition, for comparison, cells containing a connected cell aggregate formed by linking an aggregate of mesenchymal cells and an aggregate of epithelial cells were prepared using the same method except that a fiber carrier was not used. The containing gel body was similarly implanted subcutaneously into nude mice.

[result]
FIGS. 6A and 6B show photographs of a transplanted site with a cell-containing gel body attached to a fiber carrier and a transplanted site with only a cell-containing gel body not attached to a fiber carrier, 3 weeks after transplantation. are shown respectively.

As shown in FIGS. 6A and 6B, hair growth was observed at the transplanted site of the cell-containing gel body (arrow in the figure) regardless of the presence or absence of the fiber carrier. However, as shown in FIG. 6B, when only the cell-containing gel body was transplanted, the hair that grew from the transplanted site remained subcutaneously, and no hair growth was observed that penetrated the skin.

On the other hand, as shown in FIG. 6A, when the cell-containing gel body attached to the fiber carrier was transplanted, a plurality of hairs that penetrated the skin grew from the transplant site. In addition, when the cell-containing gel body attached to the fiber carrier was transplanted, the surgical tape was removed from the transplanted site on the 7th day after transplantation of the complex into a nude mouse, but the fiber carrier fell off the next day. .

[Preparation of cells]
Mesenchymal cells and epithelial cells were prepared in the same manner as in Example 1 above.

[Manufacture of composite]
A plurality of fiber carriers fixed to a support member were prepared. First, a frame-shaped support member as shown in FIGS. 2A and 3A was produced. That is, a frame-shaped PDMS support member was produced by curing PDMS into a frame shape with a square through hole formed in the center. Next, a square (3 cm x 3 cm) resin plate was placed in the central through hole of this support member. The surface of this plate was water repellent.

Thereafter, four pairs of incisions are formed in the pair of opposing fixing parts of the support member, and a medical suture thread (about 50 μm in diameter, made of nylon, manufactured by Natsume Seisakusho Co., Ltd.) with a length of about 70 mm is inserted into each pair of incisions. Both ends of one fiber carrier were inserted and fixed. Note that the free end of the fiber carrier protruding outside the support member was fixed to the back side of the support member. Thus, four fiber carriers arranged in parallel were provided, each end fixed to a support member and each central part placed on a water-repellent surface.

Next, 2 μL of the mesenchymal cell suspension was dropped in air at four locations on each fiber carrier placed on the water-repellent surface, and for each fiber carrier, collagen and dispersed mesenchymal cells were added to each fiber carrier. Four first droplets containing cells were formed, spaced apart from each other and attached to one fiber carrier.

Thereafter, 2 μL of an epithelial cell suspension was dropped in the air at a position connected to each first droplet of each fiber carrier placed on the water-repellent surface, and for each fiber carrier, collagen and Four second droplets containing dispersed epithelial cells were connected to one first droplet and spaced from each other and attached to one fiber carrier.

Thus, for each fiber carrier, there are four droplet connections, each formed by the connection of one first droplet and one second droplet, spaced apart from each other and attached to one fiber carrier. I got a body.

Further, the connected droplets attached to each fiber carrier fixed to the support member were incubated on the water-repellent surface at 37° C. for 30 minutes to gel them.

In this way, four composites were obtained, each comprising one fiber carrier fixed to a support member and four cell-containing gel bodies attached to the one fiber carrier at a distance from each other.

Thereafter, each fiber carrier and the cell-containing gel body attached to the fiber carrier were detached from the water-repellent surface. That is, four complexes on a water-repellent surface were immersed in a culture solution, and the fiber carrier and cell-containing gel body of each complex were detached from the water-repellent surface using the tip of a pipette tip.

Next, by sliding both ends of each fiber carrier to which the four cell-containing gels are attached upward along the notch in the support member, each fiber carrier floats in the culture medium while being fixed to the support member. It was kept in the same position. In this way, the cell-containing gel bodies attached to each fiber carrier were maintained in a floating state in the culture solution.

Then, within the cell-containing gel body, which is attached to each fiber carrier fixed to the support member and floating in the culture medium (mixed medium (DMEM (Sigma) + KG2 + 10% fetal bovine serum + 1% penicillin)), Leaf cells and epithelial cells were cultured for 3 days.

[result]
FIG. 7 shows a photograph of a cell-containing gel body formed in air on the surface of a water-repellent base material and attached to a fiber carrier fixed to a support member. As shown in FIG. 7, four fiber carriers were fixed to the support member, and four cell-containing gel bodies were fixed to each fiber carrier.

FIGS. 8A and 8B show optical micrographs of the complex on the first day of culture (Day 1) and the third day of culture (Day 3), respectively. As shown in FIG. 8A, each complex on the first day of culture includes one fiber carrier, each of dispersed mesenchymal cells and dispersed epithelial cells, and is spaced apart from each other and contains the one fiber carrier. and a plurality of cell-containing gel bodies attached to a fiber carrier.

On the other hand, as shown in FIG. 8B, each complex on the 3rd day of culture includes one fiber carrier, an aggregate of mesenchymal cells and an aggregate of epithelial cells that are connected to each other. It had a plurality of cell-containing gel bodies spaced apart and attached to the one fiber carrier.

Furthermore, after 3 days of culture, the cell-containing gel body attached to the fiber carrier contracted significantly. That is, the volume of the cell-containing gel body of the complex on the third day of culture was significantly decreased compared to the volume of the cell-containing gel body of the complex at the start of culture and on the first day of culture.

FIGS. 9A and 9B show phase contrast micrographs of the complex immediately after the start of culture (Day 0) and on the third day of culture (Day 3), respectively. FIGS. 10A and 10B show fluorescence micrographs of the complex immediately after the start of culture (Day 0) and on the third day of culture (Day 3), respectively.

Note that FIGS. 10A and 10B correspond to FIGS. 9A and 9B, respectively. In addition, in FIGS. 10A and 10B, mesenchymal cells are stained with the first fluorescent reagent (Vybrant™ DiI Cell-Labeling Solution, Invitrogen™) and detected as red fluorescence, and epithelial cells are detected as red fluorescence. It was stained with a second fluorescent reagent (Vybrant™ DiO Cell-Labeling Solution, Invitrogen™) and detected as green fluorescence.

As shown in FIGS. 9A and 10A, the complex immediately after the start of culture includes one fiber carrier, dispersed mesenchymal cells, and dispersed epithelial cells, which are attached to the one fiber carrier. It had a cell-containing gel body.

On the other hand, as shown in FIGS. 9B and 10B, the complex on the third day of culture consists of one fiber carrier, an aggregate of mesenchymal cells attached to the one fiber carrier, and an epithelial cell aggregate and connected to each other. and a cell-containing gel body containing aggregates of.

In addition, the aggregates of mesenchymal cells and epithelial cells covered part of the outer periphery of the fiber carrier. That is, in the composite, the fiber carrier extended to penetrate the mesenchymal cell aggregate and the epithelial cell aggregate.

Furthermore, after 3 days of culture, the cell-containing gel body attached to the fiber carrier contracted significantly. That is, the volume of the cell-containing gel body on the third day of culture was significantly reduced compared to the volume of the cell-containing gel body at the start of culture.

Claims (16)

(1) providing a substrate and a fiber carrier disposed on the surface of the substrate;
(2) forming droplets on the surface of the substrate in a gas phase that adhere to the fiber carrier and include hydrogel polymers and dispersed cells;
(3) The droplets are gelled on the surface of the base material in a gas phase to form a fiber carrier-cell-containing gel composite comprising the fiber carrier and a cell-containing gel body attached to the fiber carrier. to obtain;,
A method for producing a fiber carrier-cell-containing gel composite comprising: further comprising detaching the cell-containing gel body of the fiber carrier-cell-containing gel composite from the surface of the base material.
A method for producing the fiber carrier-cell-containing gel composite according to claim 1. further comprising culturing the cells within the cell-containing gel body of the fiber carrier-cell-containing gel complex;
A method for producing a fiber carrier-cell-containing gel composite according to claim 1 or 2. culturing the cells in the cell-containing gel body to form aggregates of the cells;
A method for producing a fiber carrier-cell-containing gel composite according to claim 3. In the above (2), on the surface of the base material in the gas phase, a plurality of fibers are attached to one of the fiber carriers at a distance from each other, each containing the hydrogel polymer and the dispersed cells. forming the droplet;
In the above (3), the droplets are gelled on the surface of the base material in a gas phase to form the one fiber carrier and a plurality of the droplets that are spaced apart from each other and adhered to the one fiber carrier. obtaining the fiber carrier-cell-containing gel composite containing a cell-containing gel body;
A method for producing a fiber carrier-cell-containing gel composite according to any one of claims 1 to 4. A fiber carrier comprising one fiber carrier piece and one cell-containing gel body attached to the one fiber carrier piece by cutting the one fiber carrier of the fiber carrier-cell-containing hydrogel composite. further comprising obtaining a piece of the cell-containing gel composite;
A method for producing a fiber carrier-cell-containing gel composite according to claim 5. In (1) above, providing a plurality of the fiber carriers arranged on the surface of the base material,
Performing (2) and (3) above for each of the plurality of fiber carriers,
A method for producing a fiber carrier-cell-containing gel composite according to any one of claims 1 to 6. In (1) above, preparing the fiber carrier in which a part is fixed to a support member and another part is disposed on the surface of the base material,
carrying out (2) and (3) above on the fiber carrier fixed to the support member;
A method for producing a fiber carrier-cell-containing gel composite according to any one of claims 1 to 7. The above (2) is
(2-1) In the gas phase, on the surface of the base material, a first droplet is formed that adheres to the fiber carrier and includes a first hydrogel polymer and dispersed first cells. to do, and
(2-2) In the gas phase, on the surface of the base material, the first droplet is attached to the fiber carrier and includes a second hydrogel polymer and dispersed second cells; forming a second droplet connected to the fiber carrier to obtain a droplet connection attached to the fiber carrier;
In the above (3), the droplet connected body is gelled on the surface of the base material in a gas phase to form a fiber carrier including the fiber carrier and the cell-containing gel body attached to the fiber carrier. - obtaining a cell-containing gel complex;
A method for producing a fiber carrier-cell-containing gel composite according to any one of claims 1 to 8. The combination of the first cell and the second cell is a combination of an epithelial cell and a mesenchymal cell,
A method for producing a fiber carrier-cell-containing gel composite according to claim 9. one fiber carrier;
a plurality of cell-containing gel bodies spaced apart from each other and attached to the one fiber carrier, each cell-containing gel body comprising a hydrogel polymer and a cell;
including,
Fiber carrier-cell-containing gel complex. Each of the plurality of cell-containing gel bodies includes aggregates of the cells,
The fiber carrier-cell-containing gel complex according to claim 11. Each of the plurality of cell-containing gel bodies includes a first gel portion containing a first hydrogel polymer and a first cell, and a second gel portion containing a second hydrogel polymer and a second cell. including
The fiber carrier-cell-containing gel complex according to claim 11 or 12. The combination of the first cell and the second cell is a combination of an epithelial cell and a mesenchymal cell,
The fiber carrier-cell-containing gel composite according to any one of claims 11 to 13. A kit for use in the method according to any one of claims 1 to 10, comprising:
a fiber carrier to which a cell-containing gel body containing a hydrogel polymer and cells is attached;
a support member to which the fiber carrier is fixed;
including,
Kit for manufacturing fiber carrier-cell-containing gel composite. The fiber carrier fixed to the support member is disposed on the surface of the fiber carrier in a gas phase, and further includes a base material on which a cell-containing gel body attached to the fiber carrier is formed.
A kit for producing a fiber carrier-cell-containing gel composite according to claim 15.

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