# US 10,865,373 B2 · Regenerated hair follicle primordium aggregation manufacturing method, hair follicle tissue-containing sheet, and method for manufacturing hair follicle tissue-containing sheet

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- Number: US 10,865,373 B2
- Document ID: US10865373B2
- Status: Granted
- Kind: B2
- Application number: 15/766,868
- Filed: 2016-10-26
- Published: 2019-02-28
- Granted: 2020-12-15
- Earliest claimed: 2015-10-30
- Assignees: National University Corporation Yokohama National University; Kanagawa Institute of Industrial Science and Technology
- Applicants: National University Corporation Yokohama National University
- Inventors: Junji Fukuda; Tatsuto Kageyama; Chisa Yoshimura; Kisaki Onishi
- Web page: https://patentgrep.com/patent/US10865373B2

## CPC

- C12M 25/04: {in combination with well or multiwell plates, i.e. culture inserts}
- A61L 27/24: Collagen
- A61L 27/362: {Skin, e.g. dermal papillae}
- A61L 27/3641: {characterised by the site of application in the body (materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60)}
- A61L 27/3813: {Epithelial cells, e.g. keratinocytes, urothelial cells}
- A61L 27/3834: {Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells}
- A61L 27/3839: {characterised by the site of application in the body (materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60)}
- A61L 27/3886: {comprising two or more cell types}
- A61L 27/52: Hydrogels or hydrocolloids
- A61L 27/60: Materials for use in artificial skin
- C12M 23/12: {Well or multiwell plates (C12M25/04 takes precedence)}
- C12N 5/0625: {Epidermal cells, skin cells; Cells of the oral mucosa}
- C12N 5/0666: {Mesenchymal stem cells from hair follicles}
- A61L 2430/18: for hair reconstruction
- A61L 2430/40: Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
- C12M 3/00: Tissue, human, animal or plant cell, or virus culture apparatus
- C12N 2500/02: Atmosphere, e.g. low oxygen conditions
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides a method for manufacturing a regular and high-density regenerated hair follicle primordium aggregation similar to the hair follicle tissue of a mammal in a simple manner. A regenerated hair follicle primordium aggregation manufacturing method of the present invention includes a step of forming hair follicle primordia by inoculating a microwell plate, which includes regularly arranged microwell portions, with mesenchymal cells and epithelial cells and culturing a mixture of the cells while supplying oxygen thereto.

## Claims

### Claim 1

A regenerated hair follicle primordium aggregation manufacturing method, comprising:
a step of inoculating a microwell plate, which has regularly arranged microwell portions, with a mixed suspension of mesenchymal cells and epithelial cells in a medium, and culturing the mixed suspension of the mesenchymal cells and the epithelial cells suspended in the medium while supplying oxygen thereto from a surface of the microwell plate so as to form the regenerated haft follicle primordia in the microwell portions, wherein the microwell plate is formed of an oxygen permeable material.

### Claim 2

A method for manufacturing a haft follicle tissue-containing sheet, comprising:
a step of inoculating a microwell plate, which has regularly arranged microwell portions, with a mixed suspension of mesenchymal cells and epithelial cells in a medium, and culturing the mixed suspension of the mesenchymal cells and the epithelial cells suspended in the medium while supplying oxygen thereto from a surface of the microwell plate so as to form the regenerated haft follicle primordia in the microwell portions; and
a step of transferring the haft follicle primordia formed in the microwell portions to a biocompatible hydrogel, wherein the microwell plate is formed of an oxygen permeable material.

### Claim 3 (depends on 2)

The method for manufacturing a hair follicle tissue-containing sheet according to claim 2, wherein a density of the microwell portions in the microwell plate is equal to or higher than 20 microwell portions/cm2 and equal to or smaller than 500 microwell portions/cm2.

### Claim 4 (depends on 1)

The regenerated hair follicle primordium aggregation manufacturing method according to claim 1, wherein the mixed suspension of the cells is cultured in a medium which does not contain a Wnt signal activator.

### Claim 5 (depends on 1)

The regenerated hair follicle primordium aggregation manufacturing method according to claim 1, wherein the mesenchymal cells and the epithelial cells are inoculated is in each of the microwell portions at the total number of 4.0×103 cells or more and 1.6×104 cells or less.

### Claim 6 (depends on 2)

The method for manufacturing a hair follicle tissue-containing sheet according to claim 2, wherein a Wnt signal activator is not used.

### Claim 7 (depends on 2)

The method for manufacturing a hair follicle tissue-containing sheet according to claim 2, wherein the total number of the mesenchymal cells and the epithelial cells with which each of the microwell portions is inoculated is equal to or greater than 4.0×103 cells and less than 1.6×104 cells.

### Claim 8 (depends on 1)

A method for treating a hair defect site caused by epidermis defect, hair loss, or the like resulting from diseases, accidents, or the like, comprising:
transplanting of an effective amount of said regenerated hair follicle primordium aggregation, which is obtained by the regenerated hair follicle primordium aggregation manufacturing method according to claim 1, to a patient in need of treatment.