Culture vessel and method for producing regenerated hair follicle primordium using the culture vessel

Abstract

To provide a culture vessel capable of simply or stably producing a regenerated hair follicle primordium, and a method for manufacturing a regenerated hair follicle primordium by using the culture vessel.SOLUTION: A culture vessel 1 comprises: a first recess 10 having a first side wall part 11 and a first bottom part 12 whose diameter becomes smaller downward; and a second recess 20 which has a second side wall part 21 and a second bottom part 22 whose diameter becomes smaller downward, and is open on the first bottom part 12 of the first recess 10, where an open angle of the first bottom part 12 is larger than an open angle of the second side wall part 21. A method for manufacturing regenerated a hair follicle primordium includes the steps of: aggregating a cell population including epithelial cells 32a inside the second recess 20 of the culture vessel; aggregating a cell population including mesenchymal cells 32b inside the second recess 20; inserting a guide thread 31 into second recess 20; and culturing a cell population including the aggregated epithelial cells 32a without using a culture support and the aggregated mesenchymal cells 32b while allowing the cells to contact with each other.SELECTED DRAWING: Figure 1

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to Japanese Patent Application No. 2019-121816, filed on June 28, 2019, the entire disclosure of which is incorporated herein by reference. [Technical field]

[0002] The present disclosure relates to a culture vessel and a method for producing a regenerated hair follicle primordium using the culture vessel. [Background technology]

[0003] As a method for treating alopecia including androgenetic alopecia (AGA), congenital alopecia, scarring, thermal alopecia, and female telogen effluvium, a treatment method is known in which epithelial stem cells and mesenchymal stem cells are separated from a small amount of hair follicle organ taken from the normal scalp of the patient, a cell population consisting of these stem cells is aggregated, a guide thread is inserted into the aggregated cell population to produce a regenerated hair follicle primordium, and this regenerated hair follicle primordium is then transplanted into the patient's alopecia site (see, for example, Patent Document 1).

[0004] According to this type of hair follicle regeneration therapy, by culturing epithelial stem cells and mesenchymal stem cells isolated from the hair follicle organ, it is possible to produce a large amount of regenerated hair follicle primordia from a small sample, thereby increasing the total number of hairs (hair follicles) on the patient's scalp without increasing invasiveness. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2012 / 108069 Summary of the Invention [Problem to be solved by the invention]

[0006] A culture vessel according to an embodiment of the present disclosure is a culture vessel for attaching a guide thread, the culture vessel comprising: a first recess having a first side wall portion and a first bottom portion that is inclined so that the diameter becomes smaller toward the bottom; a second recess having a second side wall portion and a second bottom portion that is inclined so that the diameter becomes smaller toward the bottom, the second recess being open to the first bottom portion of the first recess; and an opening angle of the first bottom portion being larger than an opening angle of the second side wall portion. The second recess has a vertex of the second bottom at the center of the opening when the opening is viewed from above. , a culture vessel.

[0007] Furthermore, a method for producing a regenerated hair follicle primordium according to an embodiment of the present disclosure is a method for producing a regenerated hair follicle primordium, comprising the steps of aggregating a cell population including epithelial cells in the second recess of any of the culture vessels according to the present disclosure, aggregating a cell population including mesenchymal cells in the second recess, inserting the guide thread into the second recess, and culturing the cell population including the aggregated epithelial cells in contact with the aggregated mesenchymal cells without using a culture support. [Brief description of the drawings]

[0008] [Figure 1] 1 is a longitudinal sectional view of a culture vessel according to an embodiment of the present disclosure. [Diagram 2] FIG. 2 is a plan view of the culture vessel shown in FIG. [Diagram 3] 2 is an enlarged cross-sectional view of a portion of a second well of the culture vessel shown in FIG. 1. [Figure 4A] FIG. 2 is a schematic diagram showing the state in which regenerated hair follicle primordia are cultured in the culture vessel shown in FIG. 1. [Figure 4B] FIG. 4B is an enlarged view of a tip portion of the guide thread in FIG. 4A. [Diagram 5] FIG. 11 is an enlarged cross-sectional view of a second recess in a modified example. [Figure 6A] FIG. 2 is an explanatory diagram showing a procedure for producing a regenerated hair follicle primordium by a method for producing a regenerated hair follicle primordium according to one embodiment of the present disclosure. [Figure 6B] FIG. 2 is an explanatory diagram showing a procedure for producing a regenerated hair follicle primordium by a method for producing a regenerated hair follicle primordium according to one embodiment of the present disclosure. [Figure 6C]FIG. 2 is an explanatory diagram showing a procedure for producing a regenerated hair follicle primordium by a method for producing a regenerated hair follicle primordium according to one embodiment of the present disclosure. [Figure 7] FIG. 13 is a table showing the hole diameters and depths of second wells of the culture vessel used in the Examples. [Figure 8] FIG. 1 shows a representative photograph of a mouse regenerated hair follicle primordium produced in a culture vessel of an example, and the formation rate of the regenerated hair follicle primordium. [Figure 9] FIG. 13 shows a photograph of a representative mouse regenerated hair follicle primordium produced using culture vessel No. 3, and a photograph of hair growth after transplantation of the regenerated hair follicle primordium. [Figure 10] FIG. 13 is a photograph showing a representative mouse regenerated hair follicle primordium produced using culture dish No. 4, and a photograph showing hair growth after transplantation of the regenerated hair follicle primordium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Conventionally, a cell population of epithelial stem cells and mesenchymal stem cells separated from a hair follicle organ is aggregated to achieve high density and compartmentalization, and then a guide thread is inserted into the cell population to produce a regenerated hair follicle primordium with a guide thread. However, conventionally, the regenerated hair follicle primordium was produced manually, which resulted in a large variation in the finished regenerated hair follicle primordium and took a long time to produce.

[0010] An object of the present disclosure is to provide a culture vessel capable of easily and stably producing regenerated hair follicle primordium, and a method for producing regenerated hair follicle primordium using the culture vessel.

[0011] According to the embodiments of the present disclosure, it is possible to provide a culture vessel capable of easily or stably producing a regenerated hair follicle primordium, and a method for producing a regenerated hair follicle primordium using the culture vessel.

[0012] Hereinafter, a culture vessel and a method for producing a regenerated hair follicle primordium according to an embodiment of the present disclosure will be described in detail with reference to the drawings.

[0013] In this specification and claims, the up-down direction refers to the up-down direction when the culture vessel is in the upright position shown in FIG.

[0014] 1 to 3, a culture vessel 1 according to this embodiment includes a first well 10 and a second well 20. The first well 10 and the second well 20 each constitute a storage space 2 of the culture vessel 1.

[0015] The storage space 2 can be configured to be liquid-tightly closed by a closing member such as a lid that is detachably attached to an opening facing upward of the culture vessel 1 (first well 10), for example.

[0016] The first recess 10 includes a first sidewall 11 and a first bottom 12. The first sidewall 11 has a cylindrical shape, and the diameter (inner diameter) is constant at each portion in the vertical direction. The first bottom 12 has a truncated cone shape that slopes downward so that the diameter (inner diameter) becomes smaller, and is integrally connected to the lower end of the first sidewall 11. The vertical length of the first bottom 12 is longer than the vertical length of the first sidewall 11. The first sidewall 11 may also be inclined.

[0017] The vertical length (depth) of the first recess 10 can be 7 to 11 mm, the vertical length (depth) of the first sidewall 11 can be 4 to 6 mm, and the vertical length (depth) of the first bottom 12 can be 3 to 5 mm. In this embodiment, the vertical length (depth) of the first recess 10 is 9 mm, the vertical length (depth) of the first sidewall 11 is 5.1 mm, and the vertical length (depth) of the first bottom 12 is 3.9 mm. The diameter of the first sidewall 11 can be 4.0 to 5.5 mm, and is 4.9 mm in this embodiment. The opening angle θ1 of the first bottom 12 can be 30 to 90°, and the opening angle θ2 of the first sidewall 11 can be 0 to 10°. In this embodiment, the opening angle θ1 of the first bottom 12 is 60°, and the opening angle θ2 of the first sidewall 11 is 4°.

[0018] In this embodiment, the first recess 10 is configured to include a first side wall portion 11, but if the first bottom portion 12 is configured to be inclined so that the diameter decreases downward, the first side wall portion 11 may not be included. In this embodiment, the first bottom portion 12 is configured to be a truncated cone shape whose diameter decreases linearly at a constant rate downward, but the shape is not limited to this and can be changed in various ways, for example, to a convex or concave curved surface shape whose diameter gradually decreases downward. In particular, if the first recess 10 is configured to include a first side wall portion 11, the first bottom portion 12 may be configured to be a planar shape perpendicular to the up-down direction.

[0019] The second recess 20 includes a second sidewall 21 and a second bottom 22. The second recess 20 is provided below the first recess 10, and opens at the second sidewall 21 to the center of the first bottom 12 of the first recess 10. The length (depth) of the second recess 20 in the vertical direction is shorter (shallower) than the length (depth) of the first recess 10 in the vertical direction.

[0020] The second side wall 21 has a cylindrical shape with a constant diameter (inner diameter) at each position in the vertical direction, and the opening 23 to the first bottom 12 is circular. The second side wall 21 has an elongated shape with its vertical length A being longer than the diameter B of the opening 23.

[0021] The second bottom 22 has a conical shape that slopes downward so that the diameter becomes smaller, and is integrally connected to the lower end of the second side wall 21. An apex 24 of the second bottom 22 is located at the center of the second bottom 22 when the opening 23 of the second recess 20 is viewed from above (in a plan view). A connection 25 where the second side wall 21 and the second bottom 22 are connected is circular, and a diameter B of the connection 25 is the same as the diameter of the opening 23.

[0022] The length of the second bottom portion 22 in the up-down direction, ie, the depth C of the second bottom portion 22, is preferably smaller than the diameter B of the connection portion 25.

[0023] Moreover, the depth C of the second bottom portion 22 is preferably smaller than the length A of the second side wall portion 21 in the up-down direction.

[0024] Furthermore, the surface roughness of the second bottom portion 22 is preferably greater than the surface roughness of the second sidewall portion 21 .

[0025] The vertical length (depth) of the second recess 20 can be 0.56 to 1.70 mm, the vertical length (depth) of the second sidewall 21 can be 0.5 to 1.5 mm, and the vertical length (depth) of the second bottom 22 can be 0.06 to 0.20 mm. In this embodiment, the vertical length (depth) of the second recess 20 is 1.0 mm, the vertical length (depth) of the second sidewall 21 is 0.9 mm, and the vertical length (depth) of the second bottom 22 is 0.09 mm. The diameter B of the second sidewall 21, i.e., the connection portion 25, can be 0.3 to 1.0 mm, and is 0.42 mm in this embodiment. The opening angle θ3 of the second bottom 22 can be 60 to 170°. In this embodiment, the opening angle θ3 of the second bottom 22 is 120°.

[0026] 4, the culture container 1 can culture a regenerated hair follicle primordium 30 in the storage space 2. The regenerated hair follicle primordium 30 has a guide thread 31 and a plurality of cells 32 located at the tip of the guide thread 31.

[0027] In the above-mentioned regenerated hair follicle primordium 30, the guide thread 31 can be formed in a thread (fibrous) shape using, for example, a polymer such as nylon, a metal such as stainless steel, or chemical fibers such as carbon fiber or glass fiber. In this embodiment, the guide thread 31 is made of nylon. The guide thread 31 used is one that is thinner than the diameter of the second side wall portion 21 of the second recess 20 (diameter B of the connection portion 25) and longer than the vertical length of the second recess 20.

[0028] The length of the guide thread 31 may be 3 to 10 mm, and the diameter of the guide thread 31 may be 0.02 to 1.20 mm. In this embodiment, the length of the guide thread 31 is 5 mm, and the diameter of the guide thread 31 is 0.5 mm.

[0029] In the above-mentioned regenerated hair follicle primordium 30, the plurality of cells 32 may be composed of, for example, a cell population including a plurality of epithelial cells 32a (shown with cross-hatching in FIG. 4) and a cell population including a plurality of mesenchymal cells 32b (shown with hatching in FIG. 4). In this case, it is preferable that the cell population including the plurality of mesenchymal cells 32b is disposed closer to the tip 31a of the guide thread 31 (to the second bottom portion 22) than the cell population including the plurality of epithelial cells 32a.

[0030] "Epithelial cells" refers to cells collected from epithelial tissue and cells obtained by culturing the collected cells, and examples include cells collected from the outermost layer of the outer root sheath of the hair follicle-derived bulge region (bulge region-derived epithelial cells), cells collected from the epidermis of adult or fetal skin, epithelial cells induced from pluripotent stem cells, epithelial progenitor cells that have the potential to differentiate into epithelial stem cells that make up hair follicles and skin, and these stem cells.

[0031] The term "mesenchymal cells" refers to cells harvested from mesenchymal tissue and cells obtained by culturing the harvested cells, and examples include cells derived from the hair papilla (e.g., hair papilla cells), hair matrix cells, root sheath cells, inner root sheath cells, outer root sheath cells (excluding the cells in the outermost layer), adult or fetal skin mesenchymal cells, hair follicle mesenchymal cells induced from pluripotent stem cells, bone marrow cells that do not contain blood cells, bone marrow-derived mesenchymal cells, bone marrow cells inside the jawbone, mesenchymal cells derived from cranial neural crest cells, or mesenchymal precursor cells or stem cells that have the potential to differentiate into these cells.

[0032] "Pluripotent stem cells" refers to cells that have the ability to differentiate into various tissues in the body, and examples of such cells include embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), and induced pluripotent stem cells (iPS cells).

[0033] The epithelial cells and mesenchymal cells used to prepare regenerated hair follicle primordia can be collected from various animals, such as mammals such as humans, monkeys, pigs, cows, horses, mice, rats, guinea pigs, rabbits, dogs, and cats.

[0034] Epithelial cells or mesenchymal cells can be harvested by surgically harvesting tissues such as hair follicles and skin or by reacting them with enzymes such as dispase, collagenase, trypsin, etc. to separate them into epithelial tissue and mesenchymal tissue, and then reacting the separated epithelial tissue or mesenchymal tissue with enzymes such as dispase, collagenase, trypsin, etc. The reaction time of the enzyme is appropriately changed depending on the size of the tissue, the properties of the cells that constitute the tissue (e.g., proteins involved in intercellular junctions, the strength of intercellular junctions, etc.), etc. In addition, two or more types of enzymes may be mixed to be reacted to separate the epithelial tissue and mesenchymal tissue or to harvest epithelial cells or mesenchymal cells from the epithelial tissue or mesenchymal tissue.

[0035] The term "cell population" refers to a cell suspension in which cells are dispersed in a liquid, and can be prepared by adding a medium capable of culturing harvested epithelial cells or mesenchymal cells, or cultured cells of these cells, and dispersing the cells by mixing.

[0036] The cells can be cultured to obtain a large amount of epithelial or mesenchymal cells. When the cells are cultured, the medium used for the culture may be a basal medium used for culturing animal cells, such as Dulbecco's modified Eagle medium (DMEM) or Advanced DMEM / F12 medium. In addition, cell growth factors such as serum, FGF (fibroblast growth factor), and EGF (epidermal growth factor) can be added to efficiently grow the epithelial or mesenchymal cells. In addition, Y27632, Noggin, SAG, etc. may be added as an additive to maintain the function of the cells, and antibiotics such as Penicillin and Streptomycin may be added as necessary. The basal medium, serum, cell growth factor, and additives are appropriately changed depending on the properties of the epithelial or mesenchymal cells. The cell culture conditions may be an environment in which the cells can grow, for example, culture in an incubator at a temperature of 37°C and a concentration of 5% CO2 is applied.

[0037] As the cell population containing a plurality of epithelial cells 32a, a cell population containing cells derived from the bulge epithelium is preferably used, and as the cell population containing a plurality of mesenchymal cells 32b, a cell population containing cells derived from the hair papilla is preferably used.

[0038] The regenerated hair follicle primordium 30 has a configuration in which a plurality of cells 32 are positioned at the tip of the guide thread 31, so that when a plurality of cells 32 are transplanted into the inside of the scalp through an incision made in the scalp, the guide thread 31 can be grasped with tweezers, making it easy to handle during transplantation. In addition, by transplanting a plurality of cells 32 into the scalp so that the guide thread 31 protrudes outside the scalp, when the plurality of cells 32 are composed of a cell population including a plurality of epithelial cells 32a and a cell population including a plurality of mesenchymal cells 32b, the plurality of cells 32 can be easily transplanted so that the cell population including the epithelial cells 32a is located on the surface side of the scalp and the cell population including the mesenchymal cells 32b is located on the inside side of the scalp. Furthermore, the guide thread 31 promotes hair growth by improving the continuity between the epithelial cells of the scalp skin tissue after transplantation and the epithelial cells 32a constituting the regenerated hair follicle primordium 30.

[0039] When culturing regenerated hair follicle primordia 30 using the culture vessel 1 of this embodiment, the storage space 2 is filled with culture medium 40, multiple cells 32 are contained inside the second recess 20, and the guide thread 31 contacts the second bottom 22 at its tip 31a facing downward, and the other end portion facing upward protrudes upward from the multiple cells 32.

[0040] Here, in the culture container 1 of this embodiment, the second bottom 22 of the second recess 20 is configured to be able to position the guide thread 31 of the regenerated hair follicle primordium 30 stored in the storage space 2 at the center of the opening 23 when the opening 23 is viewed from above (in a plan view). In other words, the second bottom 22 can easily position the guide thread 31 of the regenerated hair follicle primordium 30 so that it is disposed along the axis of the second recess 20 when the regenerated hair follicle primordium 30 is stored in the storage space 2.

[0041] In this embodiment, the second bottom 22 has a conical shape that is inclined so that the diameter decreases toward the bottom, and the apex 24 located at the center when viewed from above is the deepest recessed shape toward the bottom. Therefore, when the regenerated hair follicle primordium 30 is stored in the storage space 2 of the culture container 1, the movement of the tip 31a of the guide thread 31 in the radial direction from the apex 24 is reduced, and the guide thread 31 can be easily positioned so that it is located at the center of the opening 23 when viewed from above.

[0042] As described above, in the culture vessel 1 of this embodiment, the second recess 20 has the second bottom 22 that allows the guide thread 31 to be positioned at the center of the opening 23 when the opening 23 is viewed from above, so that when a regenerated hair follicle primordium 30 having the guide thread 31 and a plurality of cells 32 located at the tip of the guide thread 31 is cultured in the storage space 2, the guide thread 31 can be positioned at the center of the second recess 20. This allows the guide thread 31 to be positioned along the axis of the second recess 20, so that the regenerated hair follicle primordium 30 can be produced easily and stably.

[0043] Furthermore, in the culture vessel 1 of this embodiment, the second bottom 22 is configured to be inclined so that the diameter becomes smaller toward the bottom. Therefore, when the guide thread 31 is inserted inside the second recess 20, the tip 31a of the guide thread 31 is guided to the center of the second bottom 22 along the inclination of the second bottom 22, making it easier to position the guide thread 31 in the center of the opening 23.

[0044] In particular, when the second bottom 22 is conical in shape as in this embodiment, when the guide thread 31 is inserted inside the second recess 20, the tip 31a of the guide thread 31 is guided by the apex 24, and the guide thread 31 can be positioned at the center of the opening 23.

[0045] In this embodiment, the second recess 20 includes the second side wall 21, but when the second bottom 22 is inclined so that the diameter decreases downward, the second recess 20 may not include the second side wall 21. In this embodiment, the second bottom 22 is in a conical shape with a diameter decreasing downward, but the shape is not limited to this, and can be changed in various ways, for example, to a convex or concave curved surface shape with a diameter gradually decreasing downward, as long as the guide thread 31 can be positioned in the center of the opening 23 when viewed from above.

[0046] In this embodiment, the first bottom 12 is configured to be inclined so that the diameter becomes smaller toward the bottom. Therefore, when the regenerated hair follicle primordium 30 is stored in the storage space 2, the tip 31a of the guide thread 31 and the multiple cells 32 are guided along the first bottom 12 to the second recess 20, making it possible to easily store the tip 31a of the guide thread 31 and the multiple cells 32 in the second recess 20.

[0047] The culture vessel 1 having the above-mentioned configuration can be made of, for example, polystyrene. In this case, the culture vessel 1 may be formed by cutting a block of polystyrene, or may be formed by injection molding of polystyrene using a mold. When the culture vessel 1 is manufactured by injection molding of polystyrene using a mold, mass production of the culture vessel 1 is easy.

[0048] The culture vessel 1 is not limited to being made of polystyrene, but may be made of other resin materials such as Teflon (registered trademark), polypropylene, polyvinyl chloride, polyethylene, cyclic polyolefin, polyethylene terephthalate, polycarbonate, polydimethylsiloxane, polymethyl methacrylate, polyarylate, polysulfone, polyethersulfone, polyetheretherketone, polyetherimide, polymethylpentene, silicone resin, acrylic, PFA, etc., and may also be made of other materials such as glass.

[0049] In the culture vessel 1 having the above-mentioned configuration, the second recess 20 has a shape in which the vertical length A of the second sidewall portion 21 is longer than the diameter B of the opening 23, so that the multiple cells 32 cultured in the second recess 20 can be cultured to have a shape suitable for hair growth. Note that the second recess 20 may have a shape in which the vertical length A of the second sidewall portion 21 is the same as the diameter B of the opening 23, or may have a shape in which the vertical length A of the second sidewall portion 21 is shorter than the diameter B of the opening 23.

[0050] In the culture vessel 1 having the above-mentioned configuration, the second recess 20 has a shape in which the vertical length A of the second sidewall 21 is longer than the depth C of the second bottom 22, so that the cells 32 cultured in the second recess 20 can be cultured so that the bottom on the side of the tip 31a of the guide thread 31 has a rounded shape suitable for hair growth. The second recess 20 may have a shape in which the vertical length A of the second sidewall 21 is the same as the depth C of the second bottom 22, or may have a shape in which the vertical length A of the second sidewall 21 is shorter than the depth C of the second bottom 22.

[0051] Furthermore, in the culture vessel 1 having the above configuration, the second recess 20 has a shape in which the depth C of the second bottom 22 is smaller than the diameter B of the connection part 25, so that the multiple cells 32 cultured in the second recess 20 can be stably cultured so that the bottom part on the side of the tip 31a of the guide thread 31 has a rounded shape suitable for hair growth. Note that the second recess 20 may have a shape in which the depth C of the second bottom 22 is the same length as the diameter B of the connection part 25, or may have a shape in which the depth C of the second bottom 22 is larger than the diameter B of the connection part 25.

[0052] Furthermore, in the culture vessel 1 configured as above, the surface roughness of the second bottom 22 is greater than the surface roughness of the second sidewall 21, so that the plurality of cells 32 can be stably cultured in the second recess 20. Note that the second recess 20 may be configured so that the surface roughness of the second bottom 22 is the same as that of the second sidewall 21, or may be configured so that the surface roughness of the second bottom 22 is smaller than that of the second sidewall 21.

[0053] In the culture vessel 1 having the above-described configuration, the first recess 10 and the second recess 20 have a shape suitable for producing a regenerated hair follicle primordium 30, so that a plurality of cells 32 can produce a regenerated hair follicle primordium 30 having a shape suitable for hair growth.

[0054] FIG. 5 is an enlarged cross-sectional view of the second recess 20 of the modified example.

[0055] As shown in FIG. 5, the second recess 20 may be configured such that the second sidewall 21 is inclined so that the diameter decreases downward. In this case, the culture vessel 1 is configured such that the first bottom 12, the second sidewall 21, and the second bottom 22 are each inclined with respect to the vertical direction, and the angles of the first bottom 12 and the second bottom 22 with respect to the vertical direction are larger than the angle of the second sidewall 21 with respect to the vertical direction. The opening angle θ4 of the second sidewall 21 may be 0 to 30°. In this embodiment, the opening angle θ4 of the second sidewall 21 is 2°.

[0056] With this configuration, the tip 31a of the guide thread 31 and the multiple cells 32 are guided along the first bottom 12, which is inclined so that the diameter decreases downward, to the second recess 20, and are guided along the second side wall 21, which is inclined so that the diameter decreases downward, to the second bottom 22, so that the tip 31a of the guide thread 31 can be easily positioned in the center of the second bottom 22 and the multiple cells 32 can be easily stored inside the second recess 20.

[0057] Next, a method for producing regenerated hair follicle primordium 30 using the culture vessel 1 of this embodiment will be described with reference to FIG.

[0058] In the method for producing a regenerated hair follicle primordium of this embodiment, first, a step of aggregating a cell population containing mesenchymal cells 32b in the second well 20 of the culture vessel 1 is performed. In this step, a cell suspension 50 containing a plurality of mesenchymal cells (cell density: 1×10 5 pieces~2x10 6An appropriate amount of the solution (10 to 100 μL) is filled into the storage space 2 of the culture vessel 1, and centrifuged (at 500 to 700×g for 1 to 10 minutes) to aggregate a plurality of mesenchymal cells 32 b on the side of the second bottom 22 of the second well 20 (cell number: 1×10 3 ~2x10 5 In this step, the amount of cell suspension added may be any amount that does not overflow from the container, and may be any amount that does not cause functional impairment of the cells even under centrifugal conditions. As a result, as shown in FIG. 6A, a cell population including a plurality of mesenchymal cells 32b is aggregated inside the second recess 20 adjacent to the second bottom 22.

[0059] Next, a step of aggregating the cell population containing the epithelial cells 32a in the second well 20 of the culture vessel 1 is performed. In this step, the supernatant of the cell suspension 50 containing a plurality of mesenchymal cells is removed, and then a cell suspension 60 containing a plurality of epithelial cells (cell density: 1×10 5 ~4x10 6 An appropriate amount of the culture vessel 1 is filled (10 to 100 μL) into the storage space 2 of the culture vessel 1, and centrifuged (at 500 to 700×g for 1 to 10 minutes) to cause a plurality of epithelial cells 32a to be layered and aggregated above the mesenchymal cells 32b in the second well 20 (cell number: 1×10 3 ~4x10 5 In this step, the amount of cell suspension added may be any amount that does not overflow from the container, and may be any amount that does not cause functional impairment of the cells even under centrifugation conditions. As a result, as shown in FIG. 6B, a cell population including a plurality of epithelial cells 32a is aggregated inside the second well 20, overlapping above the mesenchymal cells 32b. The supernatant of the cell suspension 50 does not need to be removed.

[0060] Next, as shown in FIG. 6C, the guide thread 31 is inserted into the second recess 20. The guide thread 31 inserted into the second recess 20 penetrates the cell population consisting of the mesenchymal cells 32b and the epithelial cells 32a from above to below, and the tip 31a facing downward comes into contact with the second bottom 22. As described above, the second bottom 22 of the second recess 20 is configured to be able to position the guide thread 31 at the center of the opening 23 when the opening 23 is viewed from above, so that the guide thread 31 with the tip 31a in contact with the second bottom 22 is positioned by the second bottom 22 so that the tip 31a coincides with the apex 24, and is positioned so as to be located at the center of the opening 23 when the opening 23 is viewed from above. As a result, the guide thread 31 is positioned so as to penetrate the center of the cell population consisting of the mesenchymal cells 32b and the epithelial cells 32a.

[0061] Next, the cell population including the epithelial cells 32a aggregated in the above step and the aggregated mesenchymal cells 32b can be cultured while being in contact with each other inside the second well 20. The culture may be performed under culture conditions that allow the epithelial cells and mesenchymal cells to interact with each other while maintaining their functions, for example, for 12 to 36 hours in an incubator at a temperature of about 37°C and a concentration of 5% CO2.

[0062] By using this method for producing a regenerated hair follicle primordium, a plurality of cells 32 consisting of aggregated mesenchymal cells 32b and aggregated epithelial cells 32a are uniformly positioned around the tip of the guide thread 31, thereby easily and stably producing a regenerated hair follicle primordium 30 having a shape suitable for hair growth.

[0063] For example, by using a culture vessel 1 in which the vertical length A of the second side wall portion 21 is 900±10 μm and the diameter B of the opening 23 and connection portion 25 of the second recess 20 is 450±10 μm, it is possible to easily and stably produce the regenerated hair follicle primordium 30.

[0064] In this embodiment, the step of aggregating the cell population including mesenchymal cells 32b is performed before the step of condensing the cell population consisting of epithelial cells 32a in the second recess 20 of the culture vessel 1, but the step of aggregating the cell population including mesenchymal cells 32b may be performed after the step of condensing the cell population including epithelial cells 32a in the second recess 20 of the culture vessel 1.

[0065] In addition, in this embodiment, the step of inserting the guide thread 31 is performed after the step of aggregating the cell population including the epithelial cells 32a and the cell population including the mesenchymal cells 32b, but the step of aggregating the cell population including the epithelial cells 32a and the cell population including the mesenchymal cells 32b may be performed after first inserting the guide thread 31 into the second recess 20 and positioning the guide thread 31 at the center of the opening 23 by the second bottom 22. Even in this case, it is possible to easily and accurately produce a regenerated hair follicle primordium 30 in which a cell population consisting of mesenchymal cells 32b and epithelial cells 32a is uniformly located around the tip of the guide thread 31.

[0066] In this embodiment, the cell population containing epithelial cells 32a is a cell population containing cells derived from the epithelium of the bulge region, but this is not limiting and other cell populations can be used as the cell population containing epithelial cells 32a. Also, in this embodiment, the cell population containing mesenchymal cells 32b is a cell population containing cells derived from the hair papilla, but this is not limiting and other cell populations can be used as the cell population containing mesenchymal cells 32b. EXAMPLES

[0067] The present invention will be described in more detail below with reference to examples. Note that the present invention can be embodied in various forms with various changes in the number of cells seeded, the timing of medium replacement, the culture period, the reagents used, the concentrations of the reagents, the enzyme reaction time, etc., and is not limited to the examples described herein.

[0068] As shown in Fig. 7, the culture vessels of the embodiment each have the above configuration, and No. 1 vessel has a hole diameter (diameter of the opening) of the second recess of 0.30 mm and a depth of the second recess of 0.75 mm, No. 2 vessel has a hole diameter of the second recess of 0.30 mm and a depth of the second recess of 1.00 mm, No. 3 vessel has a hole diameter of the second recess of 0.45 mm and a depth of the second recess of 0.75 mm, No. 4 vessel has a hole diameter of the second recess of 0.45 mm and a depth of the second recess of 0.90 mm, and No. 5 vessel has a hole diameter of the second recess of 0.45 mm and a depth of the second recess of 1.00 mm. In addition, for each vessel, one formed by cutting polystyrene or Teflon (registered trademark) (cut type) was prepared.

[0069] [Example 1] Preparation of regenerated hair follicle primordia using mice and evaluation of organ induction ability

[0070] (1) Preparation of reagents

[0071] (1-1) Preparation of dissection medium DMEM (manufactured by ThermoFisher Scientific) was mixed with FBS (manufactured by Biowest, final concentration: 10%), HEPES (manufactured by ThermoFisher Scientific, final concentration: 10 mM), and penicillin-streptomycin (manufactured by ThermoFisher Scientific, final concentration: 1%).

[0072] (1-2) Preparation of culture medium for mouse hair papilla cells FBS (final concentration: 10%), penicillin-streptomycin (final concentration: 1%) and bFGF (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., final concentration: 10 ng / mL) were added to DMEM and then mixed.

[0073] (1-3) Preparation of 0.05% Trypsin-EDTA The solution was prepared by diluting 0.5% Trypsin-2 g / L EDTA·4Na (Sigma-Aldrich) 10-fold with PBS(-) (Nacalai Tesque) and mixing.

[0074] (1-4) Preparation of medium for mouse hair papilla cell recovery FBS (final concentration: 10%) and penicillin-streptomycin (final concentration: 1%) were added to DMEM and then mixed.

[0075] (1-5) Preparation of isolated enzyme solution Collagenase I (Worthington, final concentration: 50 U / mL) was added to Dispase (Becton Dickinson and Company, concentration: 50 caseinolytic U / mL) and mixed.

[0076] (1-6) Preparation of single enzyme reaction solution 2.5% Trypsin (ThermoFisher Scientific) was diluted 50-fold with PBS(-) and mixed (final concentration: 0.05%).

[0077] (1-7) Preparation of NFFSE medium Advanced DMEM / F-12 (ThermoFisherScientific) was supplemented with HEPES (ThermoFisherScientific, final concentration: 10 mM), Glutamax (ThermoFisherScientific, final concentration: 1x), B27 supplement (ThermoFisherScientific, final concentration: 1x), N2 supplement (ThermoFisherScientific, final concentration: 1x), Rock inhibitor (Y27632, Fujifilm Wako Pure Chemical Industries, final concentration: 10 μM), EGF (Peprotech, final concentration: 50 ng / mL), FGF-7 (R&D Systems, final concentration: 50 ng / mL), FGF-10 (R&D Systems, final concentration: 50 ng / mL), SHH agonist (SAG, Cayman Chemical, final concentration: 50 ng / mL), and BMP. An inhibitor (Noggin, manufactured by Peprotech, 50 ng / mL) and penicillin-streptomycin (final concentration: 1%) were added and mixed.

[0078] (1-8) Preparation of HEPES HEPES (manufactured by DOJINDO (Dojindo Laboratories)) was dissolved in MilliQ water, and the pH was adjusted to 7.4 with NaOH (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) to prepare 1 M HEPES.

[0079] (1-9) Preparation of NaHCO 1M NaHCO3 was prepared by dissolving NaHCO3 (Fujifilm Wako Pure Chemical Industries, Ltd.) in MilliQ water.

[0080] (1-10) Preparation of DMEM 10xDMEM was prepared by dissolving 1 L of DMEM powder (ThermoFisherScientific) in 100 mL of MilliQ water.

[0081] (2) Culture of mouse hair papillae

[0082] (2-1) Collection and seeding of mouse hair papillae C57BL / 6 mice (SLC) or C57BL / 6-TgN (act-EGFP) mice (SLC) aged 7-8 weeks were euthanized by cervical dislocation, and the whiskers were removed using scissors. Whisker tissue was collected using scissors without damaging the hair bulb. The whisker tissue was disinfected and washed twice each with Isodine (Meiji Seika Pharma), PBS(-), and dissection medium (see above). After removing the subcutaneous tissue of the whisker tissue in the dissection medium, the whisker hair follicles were collected by pulling them out from the whisker tissue. After selecting whisker hair follicles in anagen I to IV, the upper part of the hair bulb was cut using a scalpel, and the hair papilla was extracted from the hair bulb using a 25G injection needle. The extracted hair papilla was collected in dissection medium. The collected hair papillae were seeded onto a 35 mm culture dish (manufactured by Becton Dickinson and Company) or a 60 mm culture dish (manufactured by Becton Dickinson and Company) and cultured for 6 to 8 days in mouse hair papilla cell culture medium (see above).

[0083] (2-2) Collection of mouse dermal papilla cells After 6 to 8 days of culture, the supernatant was removed and the cells were washed twice with PBS(-). After washing, 0.05% Trypsin-EDTA (see above) was added and allowed to spread throughout, followed by reaction at 37°C for 5 minutes. The reaction was stopped by adding mouse dermal papilla cell recovery medium (see above), and the cells were recovered to prepare a cell recovery solution. The cell recovery solution was centrifuged at 250 x g and 4°C for 5 minutes, the supernatant was removed, and the cells were suspended in mouse dermal papilla cell recovery medium to prepare a mouse dermal papilla cell suspension. After preparation, the cell concentration was measured. The mouse dermal papilla cell suspension was diluted with mouse dermal papilla cell recovery medium to 1 x 10 4 The concentration was adjusted to 10 to 50 μL.

[0084] (3) Culture of mouse epithelial cells

[0085] (3-1) Collection and seeding of mouse epithelial cells The ring brust of the whisker hair follicle was cut with a scalpel and the collagen sheath was removed to separate the bulge tissue. The bulge tissue was collected in a 35 mm petri dish (Becton Dickinson & Company) containing a dissection medium. The dissection medium was removed, and the separation enzyme solution (mentioned above) was added (1 mL for approximately 150 bulge tissues), and the tissue was reacted for 5 minutes in a CO2 incubator (Panasonic Healthcare) set at 37°C and 5% CO2 concentration. After the reaction, the tissue was washed twice with dissection medium, and 2 mL of dissection medium containing 35 U / mL DNase Type I (Sigma-Aldrich) was added. The mesenchymal tissue surrounding the bulge tissue was surgically removed using a 25G injection needle to collect the bulge epithelial tissue. After washing the bulge epithelial tissue twice with PBS(-), 2 mL of the unified enzyme reaction solution (see above) was added to approximately 150 pieces of bulge epithelial tissue, and the tissue was reacted for 1 hour in a CO2 incubator set at 37°C and 5% CO2 concentration. After the reaction, the enzyme reaction was stopped by adding 1 mL of dissection medium containing 70 U / mL DNase Type I, mixed by pipetting, passed through a 35 μm cell strainer (manufactured by Corning), and centrifuged at 310 x g and 4°C for 3 minutes. After centrifugation, mouse epithelial cells were prepared by adding and mixing the dissection medium. After measuring the cell concentration of the mouse epithelial cells, the cell suspension was separated into tubes. The tube was centrifuged at 310×g for 3 minutes at 4° C. to remove the supernatant, and a 1% atelocollagen solution (Koken) containing HEPES (DOJINDO, final concentration: 10 mM), NaHCO3 (ibid., final concentration: 10 mM) and DMEM (ibid., final concentration: 1×) was added and mixed to obtain 5×10 4 ~1×10 5 A cell suspension was prepared at 100 cells / mL. After removing air bubbles by centrifugation, the cell suspension was seeded at 90 μL / well in a 6-well plate (Becton Dickinson & Company). After solidifying the gel for about 30 minutes in a CO2 incubator set at 37°C and 5% CO2 concentration, 3 mL / well of NFFSE medium was added and 3D culture was performed for 5 to 6 days in a CO2 incubator set at 37°C and 5% CO2 concentration.

[0086] (3-2) Collection of mouse epithelial cells After 5-6 days of culture, gels with cell colonies were peeled off from the culture dish with a cell scraper. The peeled off gels were collected in 1.5 mL tubes (maximum 3 gels / tube), and 1 mL of culture supernatant was added. Collagenase I was added to a final concentration of 100 U / mL, and the gel was dissolved by reacting at 37°C for 60-90 minutes. After centrifugation at 590xg and 4°C for 3 minutes, the supernatant was removed, and the gel was washed once with 1 mL of PBS(-). After washing, 500 μL of 0.125% Trypsin (ThermoFisherScientific) diluted with PBS(-) was added, and the gel was reacted at 37°C for 20 minutes. A cell suspension was prepared by adding and mixing 1 mL of dissection medium containing 35 U / mL DNase Type I. The cell suspension was passed through a 35 μm cell strainer and centrifuged at 590xg and 4°C for 3 minutes. After centrifugation, the supernatant was removed and the cells were suspended in Advanced DMEM / F-12 medium containing 1% Penicillin-Streptomycin to prepare a mouse epithelial cell suspension. After preparation, the cell concentration was measured. The mouse epithelial cell suspension was diluted to 1 × 10 4 ~2×10 4 The concentration was adjusted to 20 to 50 μL.

[0087] (4) Washing of culture vessel (vessel for reconstitution) The culture vessels of the cutting type No.1 to No.5 were immersed in 70% ethanol obtained by diluting 99.5% ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with MilliQ water, and ultrasonic waves were applied for 30 minutes at room temperature. The presence or absence of cutting residue was confirmed with a stereomicroscope (manufactured by Carl ZEISS). The culture vessels were immersed in MilliQ water (7 drops of neutral detergent were added to 50 mL of MilliQ water) to which a neutral detergent (manufactured by Kao Corporation) had been added, and washed at room temperature for 3 hours on a shaker (60 rpm). After washing, the culture vessels were washed with running water for 1 hour (the presence or absence of cutting residue was confirmed with a stereomicroscope 30 minutes after the start of washing with running water), and then immersed in MilliQ water and washed at room temperature for 1 hour on a shaker (60 rpm). Air was removed by pipetting. The MilliQ water was replaced and washed at room temperature overnight on a shaker (60 rpm). The MilliQ water was replaced again and washed at room temperature for 1 hour on a shaker (60 rpm). The plates were disinfected twice with 70% ethanol and then twice with 99.5% ethanol (air was removed by pipetting). The culture vessels were air-dried on the petri dishes in a safety cabinet (SANYO).

[0088] (6) Preparation of regenerated hair follicle primordia Regenerated hair follicle primordia were prepared using the cut-type culture vessels No. 1 to No. 5. Each washed culture vessel was inserted into a 96-well plate (Becton, Dickinson and Company). Mouse dermal papilla cell culture medium was added to the top of the second well of each culture vessel, and then 10 to 50 μL of mouse dermal papilla cell suspension was added to the culture vessel (cell number: 1 x 10 4 The cells were then centrifuged at 590 x g and room temperature for 3 to 5 minutes. After centrifugation, the supernatant was removed up to the top of the second well, and 20 to 50 μL of mouse epithelial cell suspension was added to the culture vessel (cell number: 1 × 10 4 ~2×10 4The culture vessel was then centrifuged at 590 x g for 3 to 5 minutes at room temperature (100 x 100 cells / vessel). After centrifugation, a nylon thread (Matsuda Medical Industries) with a total length of 5 mm was used as a guide thread and inserted into the second well so as to penetrate the mouse dermal papilla cells and mouse epithelial cells in the culture vessel. After insertion, the organ culture was carried out for 16 to 26 hours in a CO2 incubator set at 37°C and a CO2 concentration of 5%. After culture, the guide thread was pulled out so that the mouse dermal papilla cell aggregates and mouse epithelial cell aggregates would not come off the guide thread, and the regenerated hair follicle primordium was photographed under a phase contrast microscope (Carl ZEISS). Whether or not regenerated hair follicle primordia had been formed was determined based on the state of the mouse dermal papilla cell aggregates and mouse epithelial cell aggregates, the state of contact between the aggregates, and the positional relationship between the guide thread and each aggregate. The formation rate of regenerated hair follicle primordia (number of regenerated hair follicle primordia formed / total number of regenerated hair follicle primordia created x 100) was calculated, and the result was that a culture vessel with a hole diameter of the second recess of 0.45 mm showed a higher formation rate of regenerated hair follicle primordia, indicating that more stable regenerated hair follicle primordia can be produced (Figure 8).

[0089] (6) Evaluation of organogenesis in regenerated hair follicle primordia The regenerated hair follicle primordium was transplanted intradermally into mice according to a conventional method. That is, 6- to 8-week-old Balb / c nu / nu mice (SLC) were anesthetized according to a standard method, and the back was disinfected with isodine and placed in a natural recumbent position. A V-lance micro scalpel (Nihon Alcon) was used to puncture the skin to form a transplant wound extending from the epidermis layer to the lower dermis layer. The transplant wound was about 400 μm deep vertically from the body surface and about 1 mm horizontally. The regenerated hair follicle primordium with the guide thread inserted was inserted using sharp tweezers No. 5 (Natsume Seisakusho) so that the epithelial cell component faces the body surface side of the transplant wound. The transplant depth was adjusted so that the upper end of the epithelial cell component of the regenerated hair follicle primordium was exposed at the upper end of the transplant wound, and the nylon guide thread was positioned so that it was exposed to the body surface. The guide thread was fixed to the skin surface adjacent to the transplant wound with Steri-Strip (manufactured by 3M), and then the transplant wound was protected with Nurse-Band (manufactured by Sun Planet, registered trademark) and surgical tape (manufactured by 3M). The protective tape (Nurse-Band and surgical tape) was removed 5 to 7 days after transplantation. The survival of the graft was judged visually, and then follow-up observation (once every 2 to 4 days) was performed. For follow-up observation, the transplant site of the anesthetized mouse was observed and photographed with a fluorescent stereomicroscope (manufactured by Carl Zeiss), and the organ induction ability of the regenerated hair follicles was evaluated. The results are shown in Figures 9 and 10. The regenerated hair follicle primordia prepared in the No. 3 and No. 4 culture vessels had organ induction ability because hair grew (white arrowhead). The present disclosure is not limited to the above-described embodiment, and various modifications are possible without departing from the spirit and scope of the present disclosure. [Explanation of symbols]

[0090] 1 Culture vessel 2. Storage space 10 First recess 11 First side wall 12 1st bottom 20 Second recess 21 Second side wall 22 Second bottom 23 Aperture 24 Vertex 25 Connection 30 Regenerated hair follicle primordium 31 Guide thread 31a Tip 32 cells 32a Epithelial cells 32b Mesenchymal cells 40 Culture solution 50 Cell suspension containing multiple mesenchymal cells 60 Cell suspension containing multiple epithelial cells

Claims

1. A culture vessel for attaching a guide thread, a first recess having a first sidewall and a first bottom that slopes downward to have a smaller diameter; a second recess having a second side wall portion and a second bottom portion that is inclined so that a diameter thereof decreases downward and that opens to the first bottom portion of the first recess, an opening angle of the first bottom portion is larger than an opening angle of the second sidewall portion; A culture vessel, wherein the second well has an apex of the second bottom portion at the center of the opening when the opening is viewed from above.

2. The culture vessel according to claim 1 , A culture vessel, wherein the opening angle of the first bottom portion is 30° or more and 90° or less.

3. The culture vessel according to claim 1 or 2, A culture vessel, wherein the opening angle of the second side wall portion is equal to or greater than 0° and equal to or less than 30°.

4. The culture vessel according to any one of claims 1 to 3, The second sidewall portion extends downwardly from the first bottom portion.

5. The culture vessel according to any one of claims 1 to 4, An opening angle of the first bottom portion is larger than an opening angle of the first sidewall portion.

6. The culture vessel according to any one of claims 1 to 5, A culture vessel, wherein the opening angle of the first side wall portion is equal to or greater than 0° and equal to or less than 10°.

7. The culture vessel according to any one of claims 1 to 6, An opening angle of the second bottom portion is larger than an opening angle of the second side wall portion.

8. The culture vessel according to any one of claims 1 to 7, A culture vessel, wherein the opening angle of the second bottom portion is 60° or more and 170° or less.

9. The culture vessel according to any one of claims 1 to 8, The second bottom portion extends downwardly from the second sidewall portion.

10. The culture vessel according to any one of claims 1 to 9, A culture vessel, wherein the second bottom portion has a surface roughness greater than that of the second sidewall portion.

11. A method for producing a regenerated hair follicle primordium, comprising: A step of aggregating a cell population containing epithelial cells in the second well of the culture vessel according to any one of claims 1 to 10; Aggregating a cell population comprising mesenchymal cells in the second well; Inserting the guide thread into the second recess; and culturing a cell population containing aggregated epithelial cells without using a culture support while contacting the aggregated mesenchymal cells.

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