JP2022059393A - Methods for producing tissue body and methods for treating disease using the tissue body - Google Patents

Abstract

To provide methods for easily producing a tissue body containing a plurality of cell layers, and to provide effective methods for treating a disease using the tissue body.SOLUTION: The method for producing a tissue body comprises: a first preparation step of adding a suspension of a first cell, which is one of a mesenchymal cell and an epithelial cell, to a tubular container having a bottom; a first centrifugation step in which the tubular container containing the suspension of the first cell is subjected to a first centrifugation treatment to form a first cell layer on the bottom by centrifugal force; a second preparation step of adding a suspension of a second cell, which is the other of the mesenchymal cell and the epithelial cell, onto the first cell layer in the tubular container; a second centrifugation step in which the tubular container containing the suspension of the second cell is subjected to a second centrifugation treatment to form a second cell layer on the first cell layer by centrifugal force; and a takeout step of taking out the tissue containing the first cell layer and the second cell layer from the tubular container.SELECTED DRAWING: Figure 1E

Description

The present invention relates to a method for producing a tissue and a method for treating a disease using the tissue.

Patent Document 1 describes a method for producing an organ-type culture, which comprises culturing cells derived from an organ on a surface, in which the cells are compressed.

Patent Document 2 describes an organ-type culture apparatus, the apparatus having one open end and one end closed by a porous membrane, and the closed end is fused over the whole. A medium conduit closed by the porous membrane and a frame that holds the medium conduit substantially in the vertical direction are provided and come into contact with the surface of the porous membrane, whereby on the porous membrane. Devices to which the medium conduit is adapted are described so that sufficient volume of liquid medium to supply nutrients to the growing cells can be retained in the medium conduit by capillary phenomenon.

Patent Document 3 describes a cell addition step of adding a culture solution containing cells and cell-adhesive fine particles to a culture vessel having an inner bottom surface that is non-cell-adhesive or can be changed to non-adherent cells. The cell culture step of performing cell culture while applying a centrifugal force toward the inner bottom surface of the cells added to the culture vessel and adhering the cells to form a tissue, and the obtained cell culture step. A method for producing a tissue is described, which comprises a peeling step of peeling and collecting the tissue from the inner bottom surface.

Patent Document 4 describes a cell addition step of adding a culture solution containing cells to a culture vessel having an inner bottom surface that is non-adherent to cells or can be changed to non-adhesive cells, and to the culture vessel. A cell culture step of performing cell culture under conditions where cell-cell adhesion is formed while applying a centrifugal force toward the inner bottom surface of the added cells, and a cell culture step of forming a tissue by adhering the cells to each other, and a cell culture step. Described is a method for producing a tissue, which comprises a peeling step of peeling and recovering the tissue obtained in 1) from the inner bottom surface.

Patent Document 5 describes a method for producing a regenerative organ primordium for transplantation having a guide, which is substantially composed of a first cell aggregate substantially composed of mesenchymal cells and epithelial lineage cells. A method including a step of preparing a regenerating organ primordium by bringing the second cell aggregate into close contact and culturing inside the support and a step of inserting a guide into the regenerating organ primordium are described. ..

Japanese Patent Publication No. 2008-543303 Japanese Patent Publication No. 2008-543302 Japanese Patent Application Laid-Open No. 2015-019621 Japanese Unexamined Patent Publication No. 2010-161952 International Publication No. 2012/108069

However, conventionally, it has been difficult to easily produce an organization containing a plurality of cell layers.

The present invention has been made in view of the above problems, and the present invention is to provide a method for easily producing an organization containing a plurality of cell layers and an effective method for treating a disease using the tissue. It is one of the purposes.

In the method for producing a tissue according to an embodiment of the present invention for solving the above-mentioned problems, a suspension of first cells, which is one of mesenchymal cells and epithelial cells, is placed in a tubular container having a bottom. The first preparatory step to add, the first centrifugation step of subjecting the tubular container containing the suspension of the first cells to the first centrifugation treatment and forming the first cell layer on the bottom by centrifugal force, and the first centrifugation step. The second preparatory step of adding a suspension of the second cell, which is the other of the mesenchymal cells and the epithelial cells, and the suspension of the second cell on the first cell layer in the tubular container. The tubular container containing the tubular container is subjected to a second centrifugation treatment, and a second centrifugation step of forming a second cell layer on the first cell layer by centrifugal force, and the first cell layer and the second cell layer are included. Includes a removal step of removing the tissue from the tubular container. INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for easily producing an organization containing a plurality of cell layers.

Further, the tissue may be a hair follicle primordium. Further, the mesenchymal cells may be one or more selected from the group consisting of hair follicle mesenchymal cells and progenitor cells thereof. Further, the epithelial cells may be one or more selected from the group consisting of hair follicle epithelial cells and progenitor cells thereof.

Further, the ratio of the length of the tissue to the outer diameter of the tissue may be 1.0 or more. Further, the bottom portion of the tubular container may be impermeable. Further, the side wall portion of the tubular container may be impermeable. Further, the bottom of the tubular container may include a biocompatible layer formed of a biocompatible material, and the tissue to which the biocompatible layer is attached may be removed from the tubular container in the removal step. ..

Further, the side wall portion of the tubular container may be formed with a lubricating layer that reduces friction between the side wall portion and the tissue body when the tissue body is taken out from the tubular container in the taking-out step. In this case, the lubricating layer is composed of a material that has fluidity under the first condition and loses fluidity under the second condition, and the first preparation step and the first centrifugation step under the second condition. , The second preparation step and the second centrifugation step may be carried out, and the taking-out step may be carried out under the first condition.

Further, the centrifugation time in the first centrifugation treatment and the centrifugation time in the second centrifugation treatment may both be less than 30 minutes. Further, in the method, a fiber member is arranged in the tubular container before the take-out step to form the tissue to which the fiber member is bonded, and in the take-out step, the tubular container is used as described above. The tissue to which the fiber member is bonded may be taken out. Further, in the method, a centrifuge having a plurality of the tubular containers arranged regularly is used, and for each of the plurality of tubular containers of the centrifuge, the first preparation step, the first centrifuge step, and the like. The second preparation step, the second centrifugation step, and the taking-out step may be carried out.

In a method for treating a disease according to an embodiment of the present invention for solving the above-mentioned problems, a suspension of first cells, which is one of mesenchymal cells and epithelial cells, is added to a tubular container having a bottom. The first preparatory step, the first centrifugation step of subjecting the tubular container containing the suspension of the first cells to the first centrifugation treatment and forming the first cell layer on the bottom by centrifugal force, and the tubular. The second preparatory step of adding a suspension of the second cell, which is the other of the mesenchymal cells and the epithelial cells, onto the first cell layer in the container, and the suspension of the second cells are included. The tubular container is subjected to a second centrifugation treatment to form a second cell layer laminated on the first cell layer by centrifugal force, and includes the first cell layer and the second cell layer. It includes a step of taking out the tissue from the tubular container and a step of transplanting the tissue taken out from the tubular container into a living body having a disease. INDUSTRIAL APPLICABILITY According to the present invention, an effective method for treating a disease using a tissue is provided.

INDUSTRIAL APPLICABILITY According to the present invention, a method for easily producing an organization containing a plurality of cell layers and an effective method for treating a disease using the tissue are provided.

It is explanatory drawing which shows a part of the operation included in the example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in the example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in the example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in the example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in the example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in the example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows a part of the operation in the taking-out process included in the example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation in the taking-out process included in the example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation in the taking-out process included in the example of the manufacturing method of the structure which concerns on this Embodiment. It is explanatory drawing which shows a part of the operation included in another example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in another example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in the other example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows a part of the operation included in still another example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in still another example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in still more examples of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows a part of the operation included in still another example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in still another example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in still more examples of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows a part of the operation included in still another example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in still another example of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in still more examples of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in still more examples of the manufacturing method of the organization which concerns on this Embodiment. It is explanatory drawing which shows typically an example of the centrifuge instrument used in this embodiment. It is explanatory drawing which shows a part of the operation included in an example of the treatment method of the disease which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in the example of the treatment method of the disease which concerns on this Embodiment. It is explanatory drawing which shows the other part of the operation included in the example of the treatment method of the disease which concerns on this Embodiment. It is explanatory drawing which shows the fluorescence micrograph of an example of the hair follicle primordium produced in the Example which concerns on this Embodiment. It is explanatory drawing which shows the photograph which photographed the hair growth from the hair follicle primordium transplanted to the mouse in the Example which concerns on this Embodiment.

Hereinafter, an embodiment of the present invention will be described. The present invention is not limited to the present embodiment.

One aspect of the method according to the present embodiment (hereinafter referred to as "the present method") is a method for producing an organization. Here, in the present specification, an example of producing a hair follicle primordium for transplantation as a tissue will be mainly described.

The hair follicle primordium contains a cell layer of mesenchymal cells and a cell layer of epithelial cells, is a tissue that can be transplanted into the skin of a living body, and has the ability to grow hair in the skin of the transplanted living body. The body. The hair follicle primordium may further have the ability to grow hair in an in vitro culture system. The hair follicle primordium for transplantation is a hair follicle primordium used for transplantation into the skin of a living body.

FIGS. 1A to 1F schematically show the main steps included in this method in a cross-sectional view of the tubular container 10. It should be noted that the drawings of the present application are only schematic explanatory views, and the relative sizes of the illustrated members, cells, cell layers and the like do not always match the actual ones. Further, in the present specification, among the directions along the longitudinal direction of the tubular container 10, the direction in which the centrifugal force is applied in the centrifugal treatment (the direction indicated by the arrow X in FIG. 1A) is referred to as "downward", and the opposite direction. Is called "upward".

In this method, the first preparation step (FIG. 1B) of adding the suspension S1 of the first cell 21 which is one of the mesenchymal cells and the epithelial cells to the tubular container 10 having the bottom 11 (FIG. 1A). First centrifugation step (FIG. 1C) in which the tubular container 10 containing the suspension S1 of the first cells 21 is subjected to the first centrifugation treatment, and the first cell layer 31 is formed on the bottom 11 by centrifugal force (FIG. 1C). And the second preparation step (FIG. 1D) in which the suspension S2 of the second cell 22 which is the other of the mesenchymal cells and the epithelial cells is added onto the first cell layer 31 in the tubular container 10. The second centrifugation step of subjecting the tubular container 10 containing the suspension S2 of the second cell 22 to the second centrifugation and forming the second cell layer 32 on the first cell layer 31 by centrifugal force. (FIG. 1E) and a step (FIG. 1F) of taking out the hair follicle primordia 30 containing the first cell layer 31 and the second cell layer 32 from the tubular container 10.

In this method, mesenchymal cells and epithelial cells are used as the cells forming the hair follicle primordium 30. The first cell 21 is one of the mesenchymal cells and the epithelial cells, and the second cell 22 is the other of the mesenchymal cells and the epithelial cells. That is, when the first cell 21 is a mesenchymal cell, the second cell 22 is an epithelial cell. When the first cell 21 is an epithelial cell, the second cell 22 is a mesenchymal cell. In the production of the hair follicle primordium 30, it is preferable that the first cell 21 is a mesenchymal cell and the second cell 22 is an epithelial cell.

The mesenchymal cell is not particularly limited as long as it is a mesenchymal cell that contributes to the formation of the hair follicle primordia 30, but is one or more selected from the group consisting of the hair follicle mesenchymal cell and its progenitor cell. Is preferable.

Hair follicle mesenchymal cells are mesenchymal cells that contribute to hair growth in the hair follicle primordia (more specifically, for example, mesenchymal cells that contribute to hair growth in cooperation with hair follicle epithelial cells). Is. The hair follicle mesenchymal cells may be, for example, those collected from the hair follicles of a living body.

Further, the hair follicle mesenchymal cells may be, for example, those induced to differentiate from undifferentiated cells in vitro. The undifferentiated cells used for inducing the differentiation of hair follicle mesenchymal cells are not particularly limited as long as they have the ability to differentiate into the hair follicle mesenchymal cells, but for example, pluripotent stem cells (for example, iPS). It may be (induced Pluripotent Stem) cells, ES (Embryonic Stem) cells, or EG (Embryonic Germ) cells, or stem cells other than the pluripotent stem cells (eg, obtained by reprogramming differentiated cells). It may be a stem cell).

Specifically, the hair follicle mesenchymal cells are preferably one or more selected from the group consisting of dermal papilla cells and dermal root sheath cells, and are dermal papilla cells. Is particularly preferred.

The precursor cells of the hair follicle mesenchymal cells are not particularly limited as long as they have the ability to differentiate into the hair follicle mesenchymal cells in the hair follicle primordia, and are, for example, fetal or neonatal skin mesenchymal cells. (For example, mesenchymal cells derived from the dermal layer of fetal or neonatal skin) and one or more selected from the group consisting of stem cells that differentiate into the mesenchymal cells of the hair follicle primordia. Is preferable, and it is particularly preferable to use fetal or neonatal cutaneous mesenchymal cells.

The epithelial cell is not particularly limited as long as it is an epithelial cell that contributes to the formation of the hair follicle primordia 30, but it is preferably one or more selected from the group consisting of the hair follicle epithelial cell and its progenitor cell. ..

Hair follicle epithelial cells are epithelial cells that contribute to hair growth in the hair follicle primordia (more specifically, for example, epithelial cells that contribute to hair growth in cooperation with hair follicle mesenchymal cells). .. Hair follicle epithelial cells may be those collected from living hair follicles.

Further, the hair follicle epithelial cells may be, for example, those induced to differentiate from undifferentiated cells in vitro. The undifferentiated cells used for inducing the differentiation of hair follicle epithelial cells are not particularly limited as long as they have the ability to differentiate into the hair follicle epithelial cells, and are, for example, pluripotent stem cells (eg, iPS cells, etc.). It may be an ES cell or an EG cell), or it may be a stem cell other than the pluripotent stem cell (for example, a stem cell obtained by reprogramming a differentiated cell).

Specifically, the hair follicle epithelial lineage cells are selected from the group consisting of hair follicle epithelial stem cells, hair matrix cells, outer root shear cells, and inner root shear cells. It is preferably 1 or more, and it is particularly preferable that it is 1 or more selected from the group consisting of hair follicle epithelial stem cells, hair matrix cells, and outer root sheath cells.

The precursor cell of the hair follicle epithelial cell is not particularly limited as long as it is a cell capable of differentiating into the hair follicle epithelial cell in the hair follicle primordia, and is, for example, a fetal or neonatal cutaneous epithelial cell (for example,). It is preferably one or more selected from the group consisting of epithelial cells derived from the epidermal layer of the skin of a fetal or newborn) and stem cells that differentiate into the hair follicle epithelial cells in the hair follicle primordia. Particularly preferred are neonatal cutaneous epithelial cells.

The mesenchymal cells and epithelial cells are not particularly limited as long as they are derived from an animal having a hair follicle. The animal may be a human or a non-human animal (non-human animal). The non-human animal is preferably a non-human mammal. Non-human mammals are, for example, primates (eg, monkeys), rodents (eg, mice, rats, hamsters, guinea pigs, rabbits), meats (eg, dogs, cats), or ungulates (eg, pigs). , Cow, horse, goat, sheep). However, when producing the hair follicle primordium 30 to be transplanted into humans, human mesenchymal cells and human epithelial cells are used.

The mesenchymal cells and epithelial cells may be derived from an individual to which the hair follicle primordium 30 is transplanted, or may be derived from an individual other than the transplanted individual. For example, human mesenchymal cells and human epithelial cells are preferably derived from a patient to be transplanted, but are derived from a human other than the patient (for example, pluripotency derived from a human other than the patient). It may be a sex stem cell (for example, an iPS cell stored in a cell bank) that has been induced to differentiate.

The mesenchymal cells and / or the epithelial cells (that is, one or both of the mesenchymal cells and the epithelial cells) may be previously coated with a cell adhesion polymer. In this case, the mesenchymal cells and / or the epithelial cells have a cell-adherent polymer coating on the individual cell surface.

The cell adhesion polymer is not particularly limited as long as it is a molecule that exhibits cell adhesion to mesenchymal cells and / or epithelial cells, and is, for example, a molecule on the cell surface (for example, a protein such as integrin, or a sugar chain. ), It is preferable that it is a molecule that binds to.

Specifically, the cell adhesion polymer is, for example, a specific amino acid sequence that binds to a molecule on the cell surface (for example, a cell adhesion amino acid sequence such as arginine-glycine-aspartic acid (RGD) sequence) and / or a sugar chain. It is preferable that the polymer has.

More specifically, the cell adhesion polymer is, for example, collagen (for example, one or more collagen selected from the group consisting of type I, type II, type III, type IV, type V, and type XI), gelatin. , Fibronectin, laminin, vitronectin, elastin, glycosaminoglycan (eg, hyaluronic acid), proteoglycan, fibron, entactin and versican.

The coating treatment of the mesenchymal cells and / or the epithelial cells with the cell adhesion polymer is performed, for example, by immersing the mesenchymal cells and / or the epithelial cells in a solution containing the cell adhesion polymer. conduct. In this case, the mesenchymal cells and / or epithelial cells after immersion in a solution containing the cell adhesion polymer are washed with a solution not containing the cell adhesion polymer, and the mesenchymal cells and / or the epithelial cells are washed. Excess cell-adhering polymers that are not adhered to the cell surface of epithelial cells may be removed.

The mesenchymal cells and / or epithelial cells have a cell-adhesive polymer coating on the cell surface, whereby the cell-cell binding in the hair follicle primordium 30 produced by this method is strengthened.

In the first preparation step, the tubular container 10 is first prepared. The tubular container 10 is a tubular hollow container. The cross-sectional shape of the tubular container 10 is not particularly limited, and may be, for example, circular, elliptical, or polygonal, but is preferably circular. As shown in FIG. 1A, the tubular container 10 has an internal space 12 for accommodating a liquid or gas. The internal space 12 is surrounded by a tubular side wall portion 13 extending in the longitudinal direction of the tubular container 10 and a bottom portion 11 that closes the lower end portion 14 of the tubular container 10.

The bottom portion 11 of the tubular container 10 is not particularly limited as long as it is a member that closes the lower end portion 14 of the tubular container 10, but is preferably impermeable. That is, the bottom 11 is, for example, in the first centrifugation treatment and the second centrifugation treatment, the suspension of the first cell 21 (hereinafter referred to as “first suspension”) S1 and the suspension of the second cell 22 (hereinafter referred to as “first suspension”). Hereinafter, it is referred to as “second suspension”) and has a non-permeable property that does not allow the water contained in S2 to permeate.

The bottom 11 may be a porous body (preferably a non-permeable porous body) or a non-porous body (preferably a non-permeable non-porous body). .. The bottom 11 is preferably translucent. The translucency of the bottom 11 facilitates, for example, confirmation of the formation of a cell layer within the tubular container 10. The material constituting the bottom 11 is not particularly limited as long as the effect of the present invention can be obtained, and may be one or more selected from the group consisting of organic materials, inorganic materials and metals.

The inner surface 11a of the bottom 11 may be cell-adhesive or non-cell-adhesive. That is, when the bottom portion 11 is peeled off from the hair follicle primordium 30 and removed in the removal step, it is preferable that the inner surface 11a of the bottom portion 11 is cell-non-adhesive.

On the other hand, as will be described later, in the removal step, when the hair follicle primordium 30 to which a part or all of the bottom portion 11 is attached is taken out from the tubular container 10, the inner surface 11a of the bottom portion 11 is cell-adhesive. It is preferable to have.

The cell-adhesive inner surface 11a preferably contains a cell-adhesive polymer. The cell adhesion polymer is a cell for cells constituting the lower end surface 30a of the hair follicle primordium 30 in contact with the inner surface 11a of the bottom 11 (in the example shown in FIG. 1E, the first cell 21 of the first cell layer 31). The cell-adhesive polymer described above is preferably used, although it is not particularly limited as long as it is a molecule exhibiting adhesiveness.

In the tubular container 10, the bottom portion 11 may be provided so as to be removable. That is, in this case, the bottom portion 11 is a member that is formed independently of the side wall portion 13 and is detachably adhered to the side wall portion 13.

Further, in the tubular container 10, the bottom portion 11 and the side wall portion 13 may be integrally formed. That is, the tubular container 10 may be an integrally molded bottomed tubular body. Further, the bottom portion 11 may be formed by deforming a part of the side wall portion 13 constituting the lower end portion 14 of the tubular container 10 so as to close the lower end portion 14.

The side wall portion 13 of the tubular container 10 is not particularly limited as long as it is a tubular member surrounding the internal space 12, but is preferably impermeable. That is, the side wall portion 13 has a non-permeable property that does not allow the water contained in the first suspension S1 and the second suspension S2 to permeate in, for example, the first centrifugation treatment and the second centrifugation treatment.

The side wall portion 13 may be a porous body (preferably a non-permeable porous body) or a non-porous body (preferably a non-permeable non-porous body). good. The side wall portion 13 preferably has translucency. Since the side wall portion 13 has translucency, for example, it becomes easy to confirm the formation of the cell layer in the tubular container 10. The material constituting the side wall portion 13 is not particularly limited as long as the effect of the present invention can be obtained, and may be one or more selected from the group consisting of organic materials, inorganic materials and metals, and synthetic resins are preferable. Used.

The inner surface 13a of the side wall portion 13 may be cell-adhesive or cell non-adhesive. When the side wall portion 13 has a cell non-adhesive inner surface 13a, for example, the hair follicle primordium 30 can be easily removed from the tubular container 10 in the removal step.

The upper end 15 of the tubular container 10 may be open or closed. That is, for example, the addition of a liquid (for example, a cell suspension) to the tubular container 10 and the removal of the liquid (for example, the supernatant after centrifugation) from the tubular container 10 are carried out at the upper end of the tubular container 10. It is preferably done through the 15 openings.

On the other hand, for example, during the centrifugation or when the hair follicle primordium 30 is taken out from the lower end 14 of the tubular container 10, the upper end portion 15 of the tubular container 10 may be closed in a non-ventilable manner or may be ventilated. May be blocked. The ventilable blockage is performed, for example, by connecting an aseptic ventilable filter to the upper end 15 of the tubular container 10.

The inner diameter D ₁₀ of the tubular container may be appropriately determined according to the size of the hair follicle primordium 30 formed in the tubular container 10, but is preferably 0.050 mm or more and 2.0 mm or less, for example. , 0.075 mm or more and 1.0 mm or less are more preferable, and 0.10 mm or more and 0.50 mm or less are particularly preferable.

The length L ₁₀ of the internal space 12 of the tubular container may be appropriately determined according to the size of the hair follicle primordium 30 formed in the tubular container 10, and is, for example, 0.050 mm or more and 20.0 mm or less. It may be 0.075 mm or more, 10.0 mm or less, 0.10 mm or more, and 5.0 mm or less.

In the first preparation step, the first suspension S1 is placed in the internal space 12 of the tubular container 10 (FIG. 1B). That is, for example, the first suspension S1 is put into the internal space 12 through the opening of the upper end portion 15 of the tubular container 10.

The first suspension S1 contains an aqueous solution and the first cells 21 dispersed in the aqueous solution. The aqueous solution in which the first cell 21 is dispersed is not particularly limited as long as the survival of the first cell 21 is maintained, and for example, a physiological saline solution (for example, a phosphate buffered physiological saline solution) or a culture medium can be used. It is preferably used.

In the present specification, the cells dispersed in the aqueous solution are cells that are not bound to other cells or are attached to other cells, but the aqueous solution is made to flow by an operation such as pipetting. This is a cell that is easily separated from the other cells.

The density of the first cells 21 in the first suspension S1 is not particularly limited as long as it is within the range in which the first cell layer 31 is formed by the first centrifugation treatment, but is, for example, 5 × 10 ³ cells / mL or more. It may be 1 × 10 ⁷ cells / mL or less, preferably 1 × 10 ⁴ cells / mL or more and 5 × 10 ⁶ cells / mL or less, and 5 × 10 ⁴ cells / mL or more and 1 × 10 It is particularly preferably ⁶ cells / mL or less.

The first suspension S1 mainly contains the first cells 21 as cells. The ratio of the number of first cells 21 contained in the first suspension S1 to the total number of cells contained in the first suspension S1 is, for example, 50% or more (50% or more, 100% or less). It may be 60% or more, more preferably 70% or more, further preferably 80% or more, and particularly preferably 90% or more. The first suspension S1 may contain components other than the first cell 21, but may not contain, for example, a hydrogel polymer at a gelable concentration.

In the first centrifugation step, the tubular container 10 containing the first suspension S1 is subjected to the first centrifugal treatment, and the first cell layer 31 is formed on the bottom 11 of the tubular container 10 by centrifugal force (FIG. 1CC). ).

The first centrifugal treatment is a treatment in which a centrifugal force is applied to the lower part of the tubular container 10. By the first centrifugation treatment, the first cells 21 suspended in the first suspension S1 in the tubular container 10 settle toward the bottom 11 of the tubular container 10. The first cells 21 deposited by sedimentation are further compressed by centrifugal force. As a result, the first cell layer 31, which is an aggregate of the first cells 21, is formed on the bottom 11.

The centrifugal force generated by the first centrifugation treatment is not particularly limited as long as it is within the range in which the first suspension S1 forms the first cell layer 31 in the tubular container 10, but is, for example, 30 G or more. It is also preferable that it is 60 G or more, more preferably 90 G or more, and particularly preferably 120 G or more. The centrifugal force generated by the first centrifugal treatment may be, for example, 2000 G or less, preferably 1000 G or less, more preferably 500 G or less, and particularly preferably 250 G or less. The centrifugal force generated in the first centrifugal treatment may be specified by arbitrarily combining any of the above lower limit values and any of the above upper limit values.

The time of the first centrifugation is not particularly limited as long as it is within the range in which the first cell layer 31 is formed from the first suspension S1 in the tubular container 10, but is, for example, 0.5 minutes or more. It may be 1 minute or more, or 2 minutes or more. Further, the time of the first centrifugation treatment may be, for example, 60 minutes or less, 45 minutes or less, or 30 minutes or less.

The time of the first centrifugation is preferably less than, for example, 30 minutes. In this case, the time of the first centrifugation may be, for example, 29 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, or 10 minutes. It may be less than a minute or less than 5 minutes. The time of the first centrifugation treatment may be specified by arbitrarily combining any of the above lower limit values and any of the above upper limit values.

In the present embodiment, the centrifugal treatment is preferably performed by installing the tubular container 10 in the centrifuge and operating the centrifuge. In this case, the centrifugal force generated by the centrifugal treatment is the centrifugal force generated by the rotation speed (maximum rotation speed in the centrifugal treatment) set by the centrifuge. The centrifuge time is the time to continue centrifuging at the rotation speed (maximum rotation speed in the centrifuge) set by the centrifuge.

The density of the first cells 21 in the first cell layer 31 may be, for example, 5 × 10 ⁵ cells / cm ³ or more, preferably 1 × 10 ⁶ cells / cm ³ or more, and 5 × 10 ⁶ It is particularly preferable that the cells / cm is ³ or more. The upper limit of the density of the first cell 21 in the first cell layer 31 is not particularly limited, but the density may be, for example, 1 × 10 ⁷ cells / cm ³ or less.

The first cell layer 31 may be in contact with the inner surface 11a of the bottom 11 of the tubular container 10. Specifically, in this case, the first cell 21 constituting the lower end surface 31a of the first cell layer 31 is in contact with the inner surface 11a of the bottom portion 11.

In the second preparation step, the second suspension S2 is placed on the first cell layer 31 in the internal space 12 of the tubular container 10 (FIG. 1D). That is, for example, the second suspension S2 is put into the internal space 12 through the opening of the upper end portion 15 of the tubular container 10 after the first centrifugation treatment.

The second suspension S2 contains an aqueous solution and the second cells 22 dispersed in the aqueous solution. The aqueous solution in which the second cell 22 is dispersed is not particularly limited as long as the survival of the second cell 22 is maintained, and for example, a physiological saline solution (for example, a phosphate buffered physiological saline solution) or a culture medium can be used. It is preferably used.

The density of the second cells 22 in the second suspension S2 is not particularly limited as long as it is within the range in which the second cell layer 32 is formed by the second centrifugation, but is, for example, 5 × 10 ³ cells / mL or more. It may be 1 × 10 ⁷ cells / mL or less, preferably 1 × 10 ⁴ cells / mL or more and 5 × 10 ⁶ cells / mL or less, and 5 × 10 ⁴ cells / mL or more and 1 × 10 It is particularly preferably ⁶ cells / mL or less.

The second suspension S2 mainly contains the second cell 22 as cells. The ratio of the number of second cells 22 contained in the second suspension S2 to the total number of cells contained in the second suspension S2 is, for example, 50% or more (50% or more, 100% or less). It may be 60% or more, more preferably 70% or more, further preferably 80% or more, and particularly preferably 90% or more. The second suspension S2 may contain the components of the second cell 22, but may, for example, be free of the hydrogel polymer at a gelable concentration.

In the second centrifugation step, the tubular container 10 containing the second suspension S2 is subjected to the second centrifugation treatment on the first cell layer 31, and the first cell layer 31 in the tubular container 10 is subjected to centrifugal force. A second cell layer 32 is formed on top (Fig. 1E).

The second centrifugal treatment is a treatment in which a centrifugal force is applied to the lower part of the tubular container 10. By the second centrifugation, the second cells 22 suspended in the second suspension S2 in the tubular container 10 settle toward the preformed first cell layer 31. The second cells 22 deposited by sedimentation are further compressed by centrifugal force. As a result, the second cell layer 32, which is an aggregate of the second cells 22, is formed on the first cell layer 31.

The centrifugal force generated by the second centrifugation is not particularly limited as long as it is within the range in which the second suspension S2 forms the second cell layer 32 in the tubular container 10, but is, for example, 30 G or more. It is also preferable that it is 60 G or more, more preferably 90 G or more, and particularly preferably 120 G or more. The centrifugal force generated by the first centrifugal treatment may be, for example, 2000 G or less, preferably 1000 G or less, more preferably 500 G or less, and particularly preferably 250 G or less. The centrifugal force generated in the first centrifugal treatment may be specified by arbitrarily combining any of the above lower limit values and any of the above upper limit values.

The time of the second centrifugation is not particularly limited as long as it is within the range in which the second cell layer 32 is formed from the second suspension S2 in the tubular container 10, but is, for example, 0.5 minutes or more. It may be 1 minute or more, or 2 minutes or more. Further, the time of the second centrifugation may be, for example, 60 minutes or less, 45 minutes or less, or 30 minutes or less.

Further, the time of the second centrifugation is preferably less than, for example, 30 minutes. In this case, the time of the second centrifugation may be, for example, 29 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, or 10 minutes. It may be less than a minute or less than 5 minutes. The time of the second centrifugation may be specified by arbitrarily combining any of the above lower limit values and any of the above upper limit values.

The density of the second cell 22 in the second cell layer 32 may be, for example, 5 × 10 ⁵ cells / cm ³ or more, preferably 1 × 10 ⁶ cells / cm ³ or more, and 5 × 10 ⁶ It is particularly preferable that the cells / cm is ³ or more. The upper limit of the density of the second cell 22 in the second cell layer 32 is not particularly limited, but the density may be, for example, 1 × 10 ⁷ cells / cm ³ or less.

In the tubular container 10, the second cell layer 32 is preferably in contact with the first cell layer 31. Specifically, in this case, at the boundary between the first cell layer 31 and the second cell layer 32, a part of the first cell 21 contained in the first cell layer 31 and the second cell layer 32 are included. It comes into contact with a part of the second cell 22.

When the first cell layer 31 and the second cell layer 32 come into contact with each other, the interaction between the first cell 21 contained in the first cell layer 31 and the second cell 22 contained in the second cell layer 32 ( For example, the interaction that contributes to hair growth from the hair follicle basal 30 as is performed in the hair follicle of a living body) is effectively achieved.

In the removal step, the hair follicle primordium 30 containing the first cell layer 31 and the second cell layer 32 is taken out from the tubular container 10 (FIG. 1F). The method for taking out the hair follicle original group 30 from the tubular container 10 is not particularly limited as long as it is a method for taking out the hair follicle original group 30 from the internal space 12 of the tubular container 10 to the outside of the tubular container 10. It is preferable to take out the hair follicle original group 30 from the lower end portion 14 of 10.

2A-2C schematically show an example of an operation of removing the hair follicle primordium 30 from the tubular container 10. In this example, first, the bottom portion 11 (FIG. 2A) of the tubular container 10 containing the hair follicle primordium 30 is removed from the tubular container 10 (FIG. 2B).

The method for removing the bottom portion 11 from the tubular container 10 is not particularly limited. For example, when the bottom portion 11 is provided so as to be removable, the bottom portion 11 is removed from the side wall portion by pulling a part of the bottom portion 11. It may be peeled off from 13.

Then, the hair follicle primordium 30 is taken out from the opening of the lower end portion 14 of the tubular container 10 formed by removing the bottom portion 11. The method for taking out the hair follicle primordia 30 from the lower end 14 of the tubular container 10 is not particularly limited, but for example, the internal space 12 above the hair follicle primordia 30 in the tubular container 10 is pressurized (more specifically). Pressurizes the liquid and / or gas contained in the internal space 12) to push out the hair follicle primordia 30 from the lower end portion 14.

The hair follicle primordium 30 taken out from the tubular container 10 has a pillar shape corresponding to the internal shape of the tubular container 10. That is, the hair follicle primordium 30 manufactured using the cylindrical tubular container 10 has a cylindrical shape.

The outer diameter D ₃₀ (FIG. 1F) of the hair follicle primordium 30 is not particularly limited as long as the effect of the present invention can be obtained, but is preferably 0.050 mm or more and 2.0 mm or less, for example. , 0.075 mm or more and 1.0 mm or less are more preferable, and 0.10 mm or more and 0.50 mm or less are particularly preferable.

From the length L ₃₀ of the hair follicle primordium 30 (that is, the upper end surface 30b of the hair follicle primordium 30 (that is, the upper end surface of the uppermost cell layer (second cell layer 32 in the example of FIG. 1F)). The length up to the end face 30a (that is, the length to the lowermost cell layer (the lower end surface of the first cell layer 31 in the example of FIG. 1F)) (FIG. 1F) is within the range where the effect of the present invention can be obtained. If there is no particular limitation, for example, it may be 0.050 mm or more and 20.0 mm or less, preferably 0.075 mm or more and 10.0 mm or less, and 0.10 mm or more and 5.0 mm or less. It is particularly preferable to have.

The ratio of the length L ₃₀ of the hair follicle primordium 30 to the outer diameter D ₃₀ of the hair follicle primordium 30 (hereinafter referred to as “L / D ratio”) is within the range in which the effect of the present invention can be obtained. Although not particularly limited, for example, it may be 0.2 or more, 0.5 or more, 0.8 or more, 1.0 or more, and 1 It may be 5.5 or more, 2.0 or more, 3.0 or more, 4.0 or more, or 5.0 or more.

The upper limit of the L / D ratio of the hair follicle primordium 30 is not particularly limited, but the length / outer diameter ratio may be, for example, 10.0 or less, or 9.0 or less. , 8.0 or less, 7.0 or less, or 6.0 or less. The L / D ratio of the hair follicle primordium 30 may be specified by arbitrarily combining any of the above lower limit values and any of the above upper limit values.

The bottom 11 of the tubular container 10 may include a biocompatible layer 111 formed of a biocompatible material. 3A-3C schematically show an example of the case where the tubular container 10 has a bottom portion 11 including a biocompatible layer 111.

The biocompatible material constituting the biocompatible layer 111 is not particularly limited as long as the effect of the present invention can be obtained, but is preferably a transplantable material and / or biodegradable.

In the examples shown in FIGS. 3A and 3B, the biocompatible layer 111 constitutes the inner surface 11a of the bottom 11. Further, in the examples shown in FIGS. 3A and 3B, the hair follicle primordium 30 is in contact with the biocompatible layer 111. That is, the cells constituting the lower end surface 30a of the hair follicle primordium 30 (in the example of FIGS. 3A and 3B, the first cells 21 included in the first cell layer 31) are the inner surface 11a composed of the biocompatible layer 111. Is in contact with.

The biocompatible layer 111 is preferably cell-adhesive. In this case, the cells constituting the lower end surface 30a of the hair follicle primordium 30 (in the example shown in FIGS. 3A and 3B, the first cell 21 contained in the first cell layer 31) adhere to the biocompatible layer 111. , The bond between the hair follicle primordium 30 and the biocompatible layer 111 is strengthened.

The cell-adhesive biocompatible layer 111 is preferably composed of a cell-adhesive polymer. The cell adhesion polymer constituting the biocompatible layer 111 has cell adhesion to the cells constituting the lower end surface 30a of the hair follicle primordia 30 in contact with the inner surface 11a of the bottom 11 composed of the biocompatible layer 111. The above-mentioned cell adhesion polymer is preferably used, although the molecule is not particularly limited as long as it is a molecule showing.

Specifically, the cell adhesion polymer constituting the biocompatible layer 111 is selected from the group consisting of, for example, collagen (for example, type I, type II, type III, type IV, type V, and type XI1). It may be one or more selected from the group consisting of collagen), gelatin, fibronectin, laminin, vitronectin, elastin, glycosaminoglycan (for example, hyaluronic acid), proteoglycan, fibron, entactin and versican.

The biocompatible layer 111 may be composed of, for example, hydrogel. The hydrogel is preferably a hydrogel containing the above-mentioned cell adhesion polymer. Specifically, the biocompatible layer 111 includes, for example, collagen (for example, one or more collagen selected from the group consisting of type I, type II, type III, type IV, type V, and type XI), gelatin, and fibronectin. , Laminin, Vitronectin, Elastin, Glycosaminoglycan (eg, hyaluronic acid), Proteoglycan, Fibrin, Entactin and Versican. It is particularly preferably composed of a hydrogel containing one or more selected from the group consisting of fibrin and a laminin-entactin complex. The hydrogel constituting the biocompatible layer 111 may be formed, for example, by chemical cross-linking or photocross-linking using artificial introduction of a cross-linking functional group into the hydrogel polymer, addition of a cross-linking agent, or the like. The biocompatible layer 111 may be, for example, a dried hydrogel. A dried hydrogel can be obtained, for example, by freeze-drying the hydrogel.

When the bottom portion 11 includes the biocompatible layer 111, the bottom portion 11 may be composed of only the biocompatible layer 111 as shown in FIG. 3A, or may be composed of the biocompatible layer 111 as shown in FIG. 3B. The 111 may include a coating layer 112 that covers the lower part of the biocompatible layer 111.

The coating layer 112 is not particularly limited as long as it is a member that covers the lower part of the biocompatible layer 111, but is preferably impermeable, for example. Further, the coating layer 112 is preferably a non-porous material. The material constituting the coating layer 112 is not particularly limited, and may be one or more selected from the group consisting of an organic material, an inorganic material, and a metal, and a synthetic resin is preferably used.

The coating layer 112 is preferably removable from the biocompatible layer 111. That is, it is preferable that the coating layer 112 is provided so as to be removable from the biocompatible layer 111 while maintaining the adhesion between the biocompatible layer 111 and the hair follicle primordium 30. By including the coating layer 112 in addition to the biocompatible layer 111, the strength of the bottom portion 11 is improved.

When the bottom portion 11 of the tubular container 10 includes the biocompatible layer 111, the hair follicle primordium 30 to which the biocompatible layer 111 is attached may be removed from the tubular container 10 as shown in FIG. 3C in the removal step.

For example, when the bottom portion 11 of the tubular container 10 is composed of only the biocompatible layer 111, first, the tubular container 10 having only the biocompatible layer 111 as the bottom portion 11 is used, and the first preparation step and the first centrifugation step are performed. , A second preparation step, and a second centrifugation step are carried out to form a hair follicle primordia 30 in the tubular container 10 (FIG. 3A).

Then, in the taking-out step, for example, by pressurizing the internal space 12 above the hair follicle primordium 30 in the tubular container 10, biocompatibility from the lower end portion 14 of the tubular container 10 as shown in FIG. 3C. The layer 111 and the hair follicle primordium 30 are integrally extruded.

Further, for example, when the bottom portion 11 of the tubular container 10 includes the biocompatible layer 111 and the coating layer 112, the first preparation is performed using the tubular container 10 having the bottom portion 11 including the biocompatible layer 111 and the coating layer 112. A step, a first centrifugation step, a second preparation step, and a second centrifugation step are carried out to form a hair follicle primordium 30 in the tubular container 10 (FIG. 3B).

Then, in the taking-out step, first, the covering layer 112, which is a part of the bottom portion 11, is removed from the tubular container 10. That is, the coating layer 112 is peeled off from the biocompatible layer 111. As a result, the tubular container 10 has only the biocompatible layer 111 as the bottom 11 as shown in FIG. 3A.

Then, for example, by pressurizing the internal space 12 above the hair follicle primordium 30 in the tubular container 10, the biocompatible layer 111 and the hair follicle are formed from the lower end portion 14 of the tubular container 10 as shown in FIG. 3C. Extrude the primordium 30 integrally.

When the bottom portion 11 of the tubular container 10 includes the biocompatible layer 111 and the coating layer 112, the lower end of the tubular container 10 is located between the coating layer 112 and the hair follicle primordium 30 in the extraction step. The covering layer 112 may be removed by cutting the portion 14.

That is, in this case, a part of the biocompatible layer 111 and a part of the side wall portion 13 of the tubular container 10 are removed together with the coating layer 112. Then, as shown in FIG. 3C, the remaining biocompatible layer 111 and the hair follicle primordium 30 are integrally extruded from the lower end portion 14 of the tubular container 10.

The biocompatible layer 111 attached to the hair follicle primordium 30 taken out from the tubular container 10 may have fluidity. That is, for example, when the biocompatible layer 111 is composed of gelatin, the hair follicle primordium 30 is formed on the biocompatible layer 111 in the tubular container 10 at a temperature at which the gelatin loses fluidity, and then the gelatin is formed. The hair follicle primordium 30 may be taken out from the tubular container 10 at a temperature at which glue has fluidity. In this case, the biocompatible layer 111 attached to the hair follicle primordium 30 taken out from the tubular container 10 is composed of fluid gelatin.

In the removal step, when the hair follicle primordium 30 to which the biocompatible layer 111 is attached is taken out from the tubular container 10, the hair follicle primordium 30 to which the biocompatible layer 111 is attached may be used as it is for transplantation to the skin of a living body. good.

That is, in this case, it can be said that this method is a method for producing a composite graft containing the hair follicle primordium 30 and the biocompatible layer 111 attached to the hair follicle primordium 30. Then, the composite graft containing the hair follicle primordium 30 and the biocompatible layer 111 is transplanted into the skin of a living body.

A lubricating layer 40 is formed on the side wall portion 13 of the tubular container 10 to reduce friction between the side wall portion 13 and the hair follicle primordium 30 when the hair follicle primordium 30 is taken out from the tubular container 10 in the removal step. May be.

4A to 4C schematically show an example of the case where the side wall portion 13 of the tubular container 10 includes the lubricating layer 40. In this example, the lubricating layer 40 constitutes the inner surface 13a of the side wall portion 13 of the tubular container 10.

The material constituting the lubricating layer 40 reduces the friction between the side wall portion 13 of the tubular container 10 and the hair follicle primordium 30 in the taking-out step as compared with the case where the side wall portion 13 does not include the lubricating layer 40. However, the material is preferably a biocompatible material. In this case, the material constituting the lubricating layer 40 is preferably a transplantable material and / or is preferably biodegradable.

Specifically, it is preferable that the lubricating layer 40 is made of, for example, a material that has fluidity under the first condition and loses fluidity under the second condition. In this case, in this method, the first preparation step, the first centrifugation step, the second preparation step, and the second centrifugation step are carried out under the second condition, and the taking-out step is carried out under the first condition.

That is, in the first preparation step, the first centrifugation step, the second preparation step, and the second centrifugation step carried out under the second condition, the lubricating layer 40 having no fluidity is the first cell layer in the tubular container 10. It functions as the inner surface 13a of the side wall portion 13 that supports the 31 and the second cell layer 32.

On the other hand, after the hair follicle primordium 30 is formed in the tubular container 10, the lubricating layer 40 acquires fluidity by switching the second condition to the first condition. Then, in the take-out step carried out under the first condition, the lubricating layer 40 having fluidity is used as a lubricating liquid for reducing the friction between the hair follicle primordium 30 moving in the tubular container 10 and the side wall portion 13. Function.

Since the side wall portion 13 of the tubular container 10 has the lubricating layer 40, for example, damage to the hair follicle primordium 30 when the hair follicle primordium 30 is taken out from the tubular container 10 can be effectively avoided.

The first condition and the second condition are not particularly limited as long as they are a combination of conditions that change the fluidity of the material constituting the lubricating layer 40, and are, for example, temperature, light, solvent composition, electric field, ionic strength, and magnetic force. It may be one or more conditions selected from the group consisting of, and it is particularly preferable that it is a temperature condition.

That is, the material constituting the lubricating layer 40 is preferably a temperature-sensitive polymer that has fluidity at the first temperature and loses fluidity at the second temperature. Specifically, the temperature-sensitive polymer is preferably one or more selected from the group consisting of, for example, gelatin, polyN-isopropylacrylamide (PNIPAM), and hydroxypropyl cellulose, and gelatin is particularly preferable. ..

In this method, a cell-free hydrogel layer 50 may be formed on the hair follicle primordium 30. 5A-5C schematically show an example of forming a hydrogel layer 50 on a hair follicle primordium 30.

In this example, first, as shown in FIG. 5A, a hair follicle primordium 30 containing a first cell layer 31 and a second cell layer 32 is formed in a tubular container 10. In the formation of the hair follicle primordium 30, the first cell layer 31 is formed using the first suspension S1 containing no hydrogel polymer at a gelable concentration, and / or the gelable concentration. The second cell layer 32 may be formed by using the second suspension S2 containing no hydrogel polymer. That is, in this case, the first cell layer 31 is not formed by embedding the first cell 21 in the hydrogel, and / or the second cell layer 32 is the first cell 22 in the hydrogel. It was not formed by embedding.

Then, as shown in FIG. 5B, the hydrogel layer 50 is formed on the hair follicle primordium 30 in the tubular container 10. That is, first, an aqueous solution containing a hydrogel polymer at a gelling concentration and containing no cells is added onto the hair follicle primordium 30 in the tubular container 10, and then the hydrogel polymer is gelled. The hydrogel layer 50 is formed on the hair follicle primordium 30. In the example shown in FIG. 5B, the hydrogel layer 50 is in contact with the upper end surface 30b of the hair follicle primordium 30.

The gelation of the hydrogel polymer in the tubular container 10 is a condition under which the hydrogel polymer gels an aqueous solution of the hydrogel polymer on the hair follicle primordium 30 (for example, the hydrogel polymer). This is done by holding at the gelling temperature).

The gelation of the hydrogel polymer is performed by subjecting the hair follicle primordium 30 to a tubular container 10 containing an aqueous solution of the hydrogel polymer under the condition that the hydrogel polymer gels. May be good.

The hydrogel polymer constituting the hydrogel layer 50 is not particularly limited as long as the effect of the present invention can be obtained, but may be, for example, the above-mentioned cell adhesion polymer. In this case, the cells constituting the upper end surface 30b of the hair follicle primordium 30 (in the example shown in FIG. 5B, the second cell 22 of the second cell layer 32) adhere to the hydrogel layer 50.

Specifically, the biocompatible layer 111 includes, for example, collagen (for example, one or more collagen selected from the group consisting of type I, type II, type III, type IV, type V, and type XI), gelatin, and fibronectin. , Laminin, vitronectin, elastin, glycosaminoglycan (eg, hyaluronic acid), proteoglycan, fibron, entactin and versican, preferably composed of a hydrogel comprising one or more selected from the group.

Then, in the taking-out step, as shown in FIG. 5C, the hair follicle primordium 30 on which the hydrogel layer 50 is laminated may be taken out from the tubular container 10. The hydrogel layer 50 laminated on the hair follicle primordium 30 contributes to, for example, maintaining the shape of the hair follicle primordium 30.

When the hair follicle primordium 30 taken out from the tubular container 10 is transplanted to the skin of a living body, the hair follicle primordium 30 on which the hydrogel layer 50 is laminated may be transplanted as it is, or the hydro The hair follicle primordium 30 from which the gel layer 50 has been removed may be transplanted.

In this method, the hair follicle primordium 30 to which the fiber portion 60 is bonded may be manufactured. That is, in this case, the hair follicle primordium 30 to which the fiber portion 60 is bound is formed in the tubular container 10, and the hair follicle primordium 30 to which the fiber portion 60 is bound is taken out from the tubular container 10.

6A to 6D schematically show an example of the hair follicle primordium 30 to which the fiber portion 60 is bonded. In the example shown in FIG. 6A, the fiber portion 60 is located on the bottom portion 11 in the tubular container 10. The lower end portion 61 of the fiber portion 60 is arranged inside the hair follicle primordium 30 (specifically, inside the first cell layer 31 and the second cell layer 32), whereby the hair follicle primordium is concerned. It is bound to the group 30. Further, the upper end portion 62 of the fiber portion 60 is a free end extending above the hair follicle primordium 30.

Then, in the taking-out step, as shown in FIG. 6B, the hair follicle primordium 30 to which the fiber portion 60 is bonded is taken out from the tubular container 10. In the hair follicle primordium 30 taken out from the tubular container 10, the fiber portion 60 extends upward from the upper end surface 30b of the hair follicle primordium 30. On the other hand, the lower end portion 61 of the fiber portion 60 may not protrude below the lower end surface 30a of the hair follicle primordium 30.

In the example shown in FIG. 6C, the lower end portion 61 of the fiber portion 60 is arranged inside the bottom portion 11 of the tubular container 10. That is, in this case, the fiber portion 60 is bonded not only to the hair follicle primordium 30 but also to the bottom portion 11 of the tubular container 10.

Specifically, in the example shown in FIG. 6C, the bottom portion 11 of the tubular container 10 includes the biocompatible layer 111, and the lower end portion 61 of the fiber portion 60 penetrates the hair follicle primordium 30 and the biocompatible layer 111. It is located inside the. That is, the fiber portion 60 is bonded not only to the hair follicle primordium 30 but also to the biocompatible layer 111 of the tubular container 10.

Then, in the taking-out step, as shown in FIG. 6D, the hair follicle primordium 30 to which the fiber portion 60 and the biocompatible layer 111 are bonded is taken out from the tubular container 10. In the hair follicle primordium 30 taken out from the tubular container 10, the fiber portion 60 extends upward from the upper end surface 30b of the hair follicle primordium 30. On the other hand, the lower end portion 61 of the fiber portion 60 extends further downward from the lower end surface 30a of the hair follicle primordium 30 and is arranged inside the biocompatible layer 111. The lower end 61 of the fiber portion 60 may not protrude below the lower end surface 111a of the biocompatible layer 111.

In the case of producing the hair follicle primordium 30 to which the fiber portion 60 is bonded, in this method, for example, the fiber portion 60 is arranged in the internal space 12 of the tubular container 10 before the first centrifugation treatment, and the fiber portion 60 is used. The hair follicle primordium 30 to which the fiber portion 60 is bonded may be formed by sequentially performing the first centrifugation treatment and the second centrifugation treatment on the arranged tubular container 10.

In this case, before the first centrifugation treatment, the lower end portion 61 of the fiber portion 60 is arranged inside the bottom portion 11 (for example, the biocompatible layer 111) of the tubular container 10, and the fiber portion 60 is fixed to the bottom portion 11. May be. By fixing the fiber portion 60 arranged in the tubular container 10 to the bottom portion 11, the position of the fiber portion 60 in the tubular container 10 can be effectively controlled.

Further, in the present method, for example, after the formation of the first cell layer 31 and before the formation of the second cell layer 32, the fiber portion 60 is arranged on the first cell layer 31 or the fiber portion. The lower end 61 of the 60 may be inserted into the first cell layer 31. In the latter case, the lower end portion 61 of the fiber portion 60 may be inserted into the bottom portion 11.

Further, in this method, for example, after the formation of the second cell layer 32, the lower end portion 61 of the fiber portion 60 may be inserted into the second cell layer 32. In this case, the lower end portion 61 of the fiber portion 60 may be inserted up to the first cell layer 31, or may be further inserted into the inside of the bottom portion 11.

The fiber portion 60 is not particularly limited as long as it is a fiber-shaped member that can be arranged in the internal space 12 of the tubular container 10, and is not limited to that shown in FIGS. 6A to 6D. The fiber portion 60 is preferably flexible. The material constituting the fiber portion 60 is not particularly limited, and any material such as an organic material, an inorganic material, a metal, and a ceramic can be used, but a biocompatible material is preferable.

The fiber portion 60 may be biodegradable or non-biodegradable. The fiber portion 60 may be a medium substance or a hollow body. The shape of the fiber portion 60 is not particularly limited, but is preferably a shape similar to, for example, animal hair (particularly human hair).

Specifically, the outer diameter D ₆₀ of the fiber portion 60 is not particularly limited as long as it is the inner diameter D ₁₀ or less of the tubular container 10, but may be, for example, 10 μm or more, 20 μm or more, or 30 μm. It may be more than 40 μm, may be 40 μm or more, and may be 50 μm or more.

The length of the fiber portion 60 is not particularly limited as long as the effect of the present invention can be obtained, but may be, for example, 0.10 mm or more and 30.0 mm or less, 0.15 mm or more and 20 mm or less. It may be 0.20 mm or more and 10.0 mm or less.

By binding the fiber portion 60 to the hair follicle primordium 30, for example, the operability of the hair follicle primordium 30 taken out from the tubular container 10 is improved. That is, for example, the operability when transferring the hair follicle primordium 30 is improved, and / or the orientation of the hair follicle primordium 30 (for example, the arrangement of the first cell layer 31 and the second cell layer 32) is grasped. Becomes easier.

When the hair follicle primordium 30 to which the fiber portion 60 is bound is transplanted to the skin of a living body, the hair follicle primordium 30 is transplanted so that the fiber portion 60 extends from the hair follicle primordium 30 to the outside of the skin. Is preferable. By transplanting the hair follicle primordium 30 to which the fiber portion 60 is bonded, a space for hair growing from the hair follicle primordium 30 after transplantation is effectively secured.

In this method, a centrifuge 70 having a plurality of regularly arranged tubular containers 10 is used, and for each of the plurality of tubular containers 10 of the centrifuge 70, a first preparation step, a first centrifuge step, and a second centrifuge step are used. The preparation step, the second centrifugation step, and the taking-out step may be carried out.

FIG. 7 schematically shows an example of the centrifuge device 70. The centrifuge 70 has a plurality of regularly arranged tubular containers 10. That is, in the centrifuge 70, the plurality of tubular containers 10 are arranged at regular intervals and are connected to each other.

Further, in the example shown in FIG. 7, the centrifuge device 70 has a reservoir portion 71 that communicates with each of the plurality of tubular containers 10 and can accommodate liquids and gases. That is, the upper end portion 15 of each tubular container 10 is open in the reservoir portion 71. Further, the upper end portion 15 of each tubular container 10 has a tapered shape whose inner diameter increases upward.

By using such a centrifuge device 70, a plurality of hair follicle primordiums 30 can be efficiently produced. That is, in the present method, in the first preparation step and the second preparation step, the first suspension S1 and the second suspension S2 are added to the reservoir portion 71 of the centrifuge device 70, respectively, so that the centrifuge device 70 can be used. The first suspension S1 and the second suspension S2 can be placed in each of the plurality of tubular containers 10.

Further, in the first centrifugation step and the second centrifugation step, by subjecting the centrifuge device 70 to the first centrifugation treatment and the second centrifugation treatment, respectively, the first cells are placed in each of the plurality of tubular containers 10 of the centrifuge device 70. 31 and the second cell layer 32 can be formed.

Another aspect of the method may be a method of treating a disease associated with hair loss. In this case, the method comprises a transplantation step of transplanting the hair follicle primordium 30 produced as described above into the skin of a living body having a disease associated with hair loss.

Specifically, in this method, for example, in addition to the first preparation step, the first centrifugation step, the second preparation step, the second centrifugation step, and the taking-out step described above, the hair follicle primordia taken out from the tubular container 10 is used. 30 may be a method for treating a disease associated with hair loss, further comprising a transplantation step of transplanting 30 to the skin of a living body having a disease associated with hair loss.

The living body into which the hair follicle primordium 30 is transplanted may be a non-human animal, but is preferably a human. The skin of the living body to which the hair follicle primordia 30 is transplanted is not particularly limited as long as the skin originally has hair, but for example, the hair follicle primordia 30 is transplanted to a human patient having a disease associated with hair loss. If so, it is preferable to transplant the hair follicle primordia 30 into the scalp of the human patient.

Diseases associated with hair loss are not particularly limited, but are, for example, androgenetic alopecia (AGA), female androgenetic alopecia (FAGA), postpartum alopecia, diffuse alopecia, and seborrhea. It consists of androgenetic alopecia, androgenetic alopecia, traction alopecia, metabolic alopecia, compression alopecia, circular alopecia, neurogenic alopecia, alopecia, systemic alopecia, and symptomatic alopecia. It may be 1 or more selected from the group.

8A-8C schematically show an example of an operation when the hair follicle primordium 30 is transplanted to the skin 200 of a living body. In this example, first, as shown in FIG. 8A, a transplanted wound 210 into which the hair follicle primordium 30 is inserted is formed in the skin 200 of a living body. The transplant wound 210 is formed, for example, by puncturing or incising the skin 200 with a medical device such as a needle or a scalpel.

Then, as shown in FIG. 8B, the lower end 14 of the tubular container 10 containing the hair follicle primordium 30 in the internal space 12 is inserted into the transplant wound 210. Then, by extruding the hair follicle primordium 30 into the transplant wound 210 from the lower end 14 of the tubular container 10, the hair follicle primordium 30 is inserted into the transplant wound 210 as shown in FIG. 8C. Thus, the hair follicle primordium 30 in the tubular container 10 is transplanted into the transplant wound 210 of the skin 200. In the examples shown in FIGS. 8A to 8C, the extraction step and the transplantation step are carried out in parallel.

In the transplantation step, the hair follicle primordium 30 (FIG. 3C) to which the biocompatible layer 111 described above is attached may be transplanted to the skin of a living body. Further, in the transplantation step, the hair follicle primordium 30 to which the biocompatible layer 111 is attached is taken out from the tubular container 10, and then the biocompatible layer 111 is removed from the hair follicle primordium 30 to remove the biocompatible layer 111. The hair follicle primordium 30 to which the hair follicle primordium 30 is not attached may be transplanted to the skin of a living body.

In the transplantation step, the hair follicle primordium 30 (FIG. 5C) on which the above-mentioned hydrogel layer 50 is laminated may be transplanted to the skin of a living body. Further, in the transplantation step, after the hair follicle primordia 30 on which the hydrogel layer 50 is laminated is taken out from the tubular container 10, the hydrogel layer 50 is removed from the hair follicle primordia 30, and the hydrogel layer is removed. The hair follicle primordia 30 on which 50 is not laminated may be transplanted to the skin of a living body.

In the transplanting step, the hair follicle primordium 30 (FIGS. 6B and 6D) to which the above-mentioned fiber portion 60 is bonded may be transplanted. In this case, in the transplanted wound 210, the fiber portion 60 bonded to the transplanted hair follicle primordium 30 extends to the outside of the skin 200 through the opening 211 of the transplanted wound 210 (that is, further above the surface 201 of the skin 200). Will be extended. As a result, the fiber portion 60 inhibits the obstruction of the opening 211 of the transplanted wound 210, thereby ensuring a space in which the hair grown from the hair follicle primordium 30 transplanted to the transplanted wound 210 extends upward.

In the transplantation step, a plurality of hair follicle primordia 30 may be transplanted to the skin 200 of a living body by using a centrifuge device 70 having a plurality of regularly arranged tubular containers 10. That is, in this case, for example, a plurality of transplant wounds 210 are formed on the skin 200 of a living body in a regular arrangement corresponding to the arrangement of the plurality of tubular containers 10 in the centrifuge 70. Then, each of the plurality of tubular containers 10 of the centrifuge 70 is inserted into one of the plurality of transplant wounds 210 as shown in FIG. 8B, and the plurality of tubular containers 10 to the plurality of transplant wounds 210. The hair follicle primordium 30 is transplanted. In this case, the regular arrangement of the plurality of transplant wounds 210 and the plurality of tubular containers 10 is preferably an arrangement corresponding to the arrangement of pores in the skin of a living body.

In this method, the hair follicle primordium 30 formed in the tubular container 10 is cooled and stored without freezing while maintaining the survival of the first cell 21 and the second cell 22 contained in the hair follicle primordium 30. It may include a cooling storage step to be performed.

In this case, in the cooling storage step, for example, the hair follicle primordium 30 may be cooled and stored in the tubular container 10. That is, for example, after the hair follicle primordium 30 is formed in the tubular container 10, and before the hair follicle primordium 30 is taken out from the tubular container 10, the hair follicle primordium 30 is cooled and stored in the tubular container 10. ..

Specifically, for example, when a plurality of hair follicle primordiums 30 are produced using a plurality of tubular containers 10, a step of taking out some of the hair follicle primordiums 30 among the plurality of hair follicle primordiums 30 is carried out. During this time, a cooling storage step is carried out for the other hair follicle primordium 30.

More specifically, for example, when a plurality of hair follicle primordia 30 produced using a plurality of tubular containers 10 are transplanted into the skin of a living body, some of the hairs of the plurality of hair follicle primordial groups 30 are transplanted. While performing the operation of transplanting the hair follicle base into the skin, the remaining hair follicle base 30 is cooled and stored in the tubular container 10.

The temperature at which the hair follicle primordium 30 is cooled and stored in the cold storage step is a low temperature at which the first cells 21 and the second cells 22 contained in the hair follicle primordium 30 can be stored without freezing while maintaining their survival. Although not particularly limited, for example, it may be in the range of more than 0 ° C. and 10 ° C. or lower, and preferably in the range of more than 0 ° C. and 5 ° C. or lower.

The method may further include a culturing step of culturing the hair follicle primordium 30 taken out of the tubular container 10. In the culturing step, for example, the hair follicle primordium 30 taken out from the tubular container 10 may be suspended-cultured in a culture solution, cultured on the surface of a substrate, or in a hydrogel. It may be embedded and cultured in.

On the other hand, in this method, the hair follicle primordium 30 taken out from the tubular container 10 may not be cultured on the surface of the substrate. Further, in this method, the hair follicle primordium 30 taken out from the tubular container 10 may not be cultured.

The hair follicle primordium 30 may further contain one or more other cells in addition to the mesenchymal cells and epithelial cells. The other cells are not particularly limited as long as they are cells that contribute to hair growth from the hair follicle primordium 30, but are preferably, for example, pigment cells and / or pigment stem cells.

One or more other cells may be included in the first cell layer 31 and / or the second cell layer 32. Specifically, for example, pigment cells and / or pigment stem cells may be contained in the first cell layer 31 and / or may be contained in the second cell layer 32, but the first cell 21 may be contained. When it is a mesenchymal cell, it is preferably contained in the first cell layer 31.

The hair follicle primordium 30 may contain only the first cell layer 31 and the second cell layer 32 as the cell layer, or one or more in addition to the first cell layer 31 and the second cell layer 32. Other cell layers may be further included.

When the hair follicle primordium 30 contains one or more other cell layers, the other cell layers are formed by the same centrifugation as the first cell layer 31 and the second cell layer 32 described above. Specifically, in this method, for example, after the first centrifugation step and before the second preparatory step, a third cell (eg, pigment cell and / or pigment) is placed on the first cell layer 31 in the tubular container 10. The third preparatory step of adding the suspension of stem cells) and the third centrifugation treatment of the tubular container 10 containing the suspension of the third cells are performed, and centrifugal force is applied onto the first cell layer 31. A third centrifugation step of forming a third cell layer was included, the first cell layer 31, the third cell layer formed on the first cell layer, and the third cell layer formed on the third cell layer. The hair follicle primordia 30 containing the second cell layer 32 may be produced.

In this method, as described above, since each cell layer contained in the hair follicle primordium 30 is formed by centrifugation, the characteristics of the hair follicle primordium 30 can be adjusted depending on the conditions of the centrifugation. That is, for example, as the centrifugation time becomes longer, the bonds between cells contained in the cell layer become stronger.

On the other hand, after transplanting the hair follicle primordium 30 into the skin of a living body, it is preferable that the cells contained in the hair follicle primordium 30 can move in the skin in order to construct a new hair follicle structure. In this respect, by shortening the centrifugation time, it is possible to prevent the bonds between the cells contained in the cell layer from becoming too strong, and it is possible to produce a hair follicle primordium containing cells that are easily migrated after transplantation. ..

Further, for transplantation for hair regeneration, it is preferable to produce thousands of hair follicle primordiums 30. In this respect, by shortening the centrifugation time, a large amount of hair follicle primordium can be rapidly produced.

Therefore, in this method, the total time of centrifugation applied to the tubular container 10 containing cells for producing the hair follicle primordium 30 (for example, after the first centrifugation treatment, 1 in addition to the second centrifugation treatment). When the above other centrifugation treatment is performed, the total of the time of the first centrifugation treatment, the time of the second centrifugation treatment, and the time of the other centrifugation treatment of 1 or more is, for example, 1 minute. It may be more than 2 minutes, it may be 2 minutes or more, and it may be 4 minutes or more.

Further, the total time of the centrifugation may be, for example, 120 minutes or less, 90 minutes or less, 60 minutes or less, or 30 minutes or less. Further, the total time of the centrifugation is preferably less than, for example, 30 minutes. In this case, the time of the first centrifugation may be, for example, 29 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, or 10 minutes. It may be less than a minute. The total time of the centrifugation may be specified by arbitrarily combining any of the above lower limit values and any of the above upper limit values.

This method is not limited to the above-mentioned example of hair follicle primordium for transplantation. That is, the hair follicle primordium produced in this method may be used for purposes other than transplantation. Specifically, the hair follicle primordium may be used, for example, for in vitro testing or research. In addition, the hair follicle primordium may be used for screening a substance related to hair growth.

In this method, a tissue other than the hair follicle primordium may be produced. The tissue body other than the hair follicle primordium may be a tissue body for transplantation or a tissue body used for a purpose other than transplantation.

When the method is a method of treating a disease, the method comprises a transplantation step of transplanting the tissue into a living body having the disease. That is, in this method, for example, in addition to the above-mentioned first preparation step, first centrifugation step, second preparation step, second centrifugation step, and removal step, the tissue taken out from the tubular container is treated with a disease. It may be a method for treating a disease, further comprising a transplantation step of transplanting into a living body having the disease.

The disease to be treated is not particularly limited as long as it is a disease having a structural and / or functional defect in the organ or tissue of the living body into which the tissue is transplanted. That is, the tissue for transplantation produced by this method is transplanted into the organ or tissue of a living body in order to restore the structure and / or function of the tissue.

The mesenchymal cell may be a mesenchymal cell derived from any organ or tissue of a living body, or may be a mesenchymal cell differentiated from a stem cell such as a pluripotent stem cell in vitro. good. The epithelial cells may be epithelial cells derived from any organ or tissue of a living body, or may be epithelial cells induced to differentiate from stem cells such as pluripotent stem cells in vitro.

When producing a tissue used for a purpose other than transplantation, the hydrogel polymer used for forming the hydrogel layer 50 described above is not limited to the above-mentioned examples, and for example, agarose, sodium alginate, and synthetic polymers ( For example, it may be one or more selected from the group consisting of polyacrylamide, polyvinyl alcohol, methyl cellulose, polyethylene oxide, etc.).

Next, a specific embodiment according to the present embodiment will be described.

[Preparation of tubular container] The opening at the tip of a commercially available resin pipette tip was closed with a silicone-based material (PDMS). The pipette tip was filled with a 20 wt% gelatin solution, and a wire having an outer diameter of about 0.3 mm and a length of about 100 mm was placed in the gelatin solution. The gelatin was then solidified by holding the pipette tip at 4 ° C. Then, by removing the wire from the pipette tip, a tubular container surrounded by gelatin and having an internal space (inner diameter of about 0.3 mm and length of about 30 mm) corresponding to the outer shape of the wire was obtained. That is, the tubular container had a bottom composed of a biocompatible layer formed of gelatin and a coating layer formed of PDMS. The tubular container also had a side wall inner surface composed of a lubricating layer made of gelatin. Then, a commercially available resin syringe was bonded to the upper end of the tubular container (that is, the upper end of the pipette tip) to prepare a centrifuge device composed of the tubular container and the syringe.

[Cell preparation] The method reported by Nakao et al. (Koh-ei Toyoshima et al. Nature Communication, Vol.3, Article Number 784, 2012) by collecting the skin tissue on the back from a C57BL / 6 mouse fetus at the age of 18 days. Was partially modified and treated with dispase at 4 ° C. for 1 time under 30 rpm shaking conditions to separate the epithelial layer and the mesenchymal layer of the skin tissue. Then, the epithelial layer was treated with 100 U / mL collagenase for 2 hours and then with trypsin for 10 minutes to finally obtain dispersed epithelial cells that had passed through a 40 μm mesh cell strainer. The mesenchymal layer was treated with 100 U / mL collagenase for 2 hours to finally obtain dispersed mesenchymal cells that had passed through a 40 μm mesh cell strainer.

[Manufacturing of hair follicle primordium]
Mesenchymal cells were stained with a red fluorescent reagent (Vybrant ™ DiI Cell-Labeling Solution, Invitrogen ™). In addition, epithelial cells were stained with a green fluorescent reagent (Vybrant ™ DiO Cell-Labeling Solution, Invitrogen ™).

Then, first, 200 μL of a suspension containing mesenchymal cells at a density of 1 × 10 ⁶ cells / mL was added to the tubular container via a syringe in the centrifuge container. Next, a centrifuge container containing a suspension of mesenchymal cells was placed in a commercially available centrifuge, and the centrifuge was subjected to centrifugal treatment at 4 ° C. at 1000 rpm (centrifugal force 180 G) for 180 seconds. As a result, the first cell layer of mesenchymal cells was formed on the biocompatible layer at the bottom of the tubular container.

Then, 200 μL of a suspension containing epithelial cells at a density of 5 × 10 ⁵ cells / mL was added to the tubular vessel via a syringe in the centrifuge vessel. Then, the centrifuge vessel containing the suspension of epithelial cells was centrifuged at 4 ° C. at 1000 rpm for 180 seconds.

As a result, a second cell layer of epithelial cells was formed in the tubular container, which was laminated on the first cell layer of mesenchymal cells. That is, a hair follicle primordium composed of a first cell layer of mesenchymal cells and a second cell layer of epithelial cells was formed.

Then, a solution containing 2.4 mg / mL of Type I collagen was added to the tubular container via a syringe in the centrifuge container. Then, the centrifuge container containing the collagen solution was centrifuged at 37 ° C. at 1000 rpm for 180 seconds.

As a result, gelatin was liquefied and collagen was gelled in the tubular container. That is, the biocompatible layer and the lubricating layer acquired fluidity in the tubular container, and a collagen hydrogel layer laminated on the hair follicle primordium was formed.

Then, the coating layer is removed by cutting the portion of the lower end of the tubular container between the coating layer and the hair follicle primordium (that is, the portion where the biocompatible layer of gelatin is arranged). An opening was formed at the lower end. Then, by pressurizing through a syringe of the centrifuge container, the hair follicle primordium in which gelatin adhered to the lower end surface and collagen gel was laminated on the upper end surface was extruded from the opening at the lower end of the tubular container.

FIG. 9 shows a fluorescence micrograph of the hair follicle primordium (the hair follicle primordium from which gelatin and collagen gel have been removed) taken out from the tubular container. In FIG. 9, the scale bar indicates 200 μm. As shown in FIG. 9, a columnar hair follicle composed of a first cell layer of mesenchymal cells stained with a red fluorescent dye and a second cell layer of epithelial cells stained with a green fluorescent dye. The primordia was formed.

[Preparation of Tubular Container] A centrifuge device including a tubular container was prepared in the same manner as in Example 1 described above.

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

[Production of hair follicle primordium] The above-mentioned is described except that the mesenchymal cells and epithelial cells were not fluorescently stained and the hydrogel layer of collagen laminated on the hair follicle primordium was not formed. Hair follicle primordium was produced in a tubular container in the same manner as in Example 1.

[Transplantation of hair follicle primordium] Nude mice were anesthetized by inhalation of isoflurane, and their backs were disinfected with isoflurane. V-lance micromes (manufactured by Nippon Archon Co., Ltd.) was used to form transplant wounds from the epidermal layer of the back skin of nude mice to the lower part of the dermis layer.

On the other hand, a portion between the PDMS coating layer of the tubular container containing the hair follicle primordium in the internal space and the hair follicle primordium was cut to form an opening at the lower end of the tubular container. Then, the lower end of the tubular container is inserted into the transplant wound of a nude mouse, and the hair follicle primordium (specifically, the hair follicle coated with fluid gelatin) is inserted into the transplant wound through the opening of the lower end. By extruding the primordium), the hair follicle primordium was transplanted into the skin of the nude mouse. Animal breeding and animal experiments were carried out in compliance with the guidelines of the Yokohama National University Animal Experiment Committee.

FIG. 10 shows a photograph obtained by photographing a portion of the skin of a nude mouse into which a hair follicle primordium was transplanted 3 weeks after transplantation. As shown in FIG. 10, it was confirmed that a plurality of hairs were grown from the transplanted hair follicle primordium.

Claims (14)

The first preparatory step of adding a suspension of the first cell, which is one of the mesenchymal cells and the epithelial cells, to the tubular container having the bottom.
A first centrifugation step in which the tubular container containing the suspension of the first cells is subjected to the first centrifugation treatment to form a first cell layer on the bottom by centrifugal force.
A second preparatory step of adding a suspension of the second cell, which is the other of the mesenchymal cells and the epithelial cells, onto the first cell layer in the tubular container.
A second centrifugation step in which the tubular container containing the suspension of the second cells is subjected to a second centrifugation treatment to form a second cell layer on the first cell layer by centrifugal force.
A step of taking out the tissue containing the first cell layer and the second cell layer from the tubular container, and
A method of manufacturing an organization, including. The tissue is a hair follicle primordium,
The method according to claim 1. The mesenchymal cell is one or more selected from the group consisting of hair follicle mesenchymal cells and progenitor cells thereof.
The method according to claim 1 or 2. The epithelial cell is one or more selected from the group consisting of hair follicle epithelial cells and progenitor cells thereof.
The method according to any one of claims 1 to 3. The ratio of the length of the tissue to the outer diameter of the tissue is 1.0 or more.
The method according to any one of claims 1 to 4. The bottom of the tubular container is impermeable,
The method according to any one of claims 1 to 5. The side wall portion of the tubular container is impermeable.
The method according to any one of claims 1 to 6. The bottom of the tubular container contains a biocompatible layer formed from a biocompatible material.
In the removal step, the tissue to which the biocompatible layer is attached is removed from the tubular container.
The method according to any one of claims 1 to 7. A lubricating layer is formed on the side wall portion of the tubular container to reduce friction between the side wall portion and the tissue body when the tissue body is taken out from the tubular container in the removal step.
The method according to any one of claims 1 to 8. The lubricating layer is composed of a material that has fluidity under the first condition and loses fluidity under the second condition.
The first preparation step, the first centrifugation step, the second preparation step, and the second centrifugation step are carried out under the second condition.
The extraction step is carried out under the first condition.
The method according to claim 9. The centrifugation time in the first centrifugation treatment and the centrifugation time in the second centrifugation treatment are both less than 30 minutes.
The method according to any one of claims 1 to 10. Prior to the removal step, a fiber member is placed in the tubular container to form the tissue to which the fiber member is bonded.
In the take-out step, the tissue to which the fiber member is bonded is taken out from the tubular container.
The method according to any one of claims 1 to 11. Using a centrifuge with the plurality of regularly arranged tubular containers,
Each of the plurality of tubular containers of the centrifuge device is subjected to the first preparation step, the first centrifugation step, the second preparation step, the second centrifugation step, and the taking-out step.
The method according to any one of claims 1 to 12. The first preparatory step of adding a suspension of the first cell, which is one of the mesenchymal cells and the epithelial cells, to a tubular container having a bottom,
A first centrifugation step in which the tubular container containing the suspension of the first cells is subjected to the first centrifugation treatment to form a first cell layer on the bottom by centrifugal force.
A second preparatory step of adding a suspension of the second cell, which is the other of the mesenchymal cells and the epithelial cells, onto the first cell layer in the tubular container.
A second centrifugation step in which the tubular container containing the suspension of the second cells is subjected to a second centrifugation to form a second cell layer laminated on the first cell layer by centrifugal force.
A step of taking out the tissue containing the first cell layer and the second cell layer from the tubular container, and
A transplantation step of transplanting the tissue taken out from the tubular container into a living body having a disease,
How to treat the disease, including.

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