# US 2019/0169558 A1 · Three-dimensional structuring method and three-dimensional structuring system of cells

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- Number: US 2019/0169558 A1
- Document ID: US20190169558A1
- Status: Published application
- Kind: A1
- Application number: 16/324,496
- Filed: 2017-06-09
- Published: 2019-06-06
- Earliest claimed: 2016-08-10
- Assignees: Cyberdyne Inc.; University of Tsukuba
- Applicants: CYBERDYNE Inc.; University of Tsukuba
- Inventors: Yoshiyuki SANKAI
- Web page: https://patentgrep.com/patent/US20190169558A1

## CPC

- C12M 25/12: {the culture medium flowing outside the fiber or tube}
- C12M 3/00: Tissue, human, animal or plant cell, or virus culture apparatus
- C12M 21/08: {for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00)}
- C12M 25/02: {Membranes; Filters (filters or filtration in general B01D24/00-B01D41/00)}
- C12M 25/10: {Hollow fibers or tubes (hollow fiber modules in general B01D63/02)}
- C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)}
- C12M 27/16: {Vibrating; Shaking; Tilting}
- C12M 29/10: {Perfusion}
- C12M 29/16: {Hollow fibers (hollow fiber modules in general B01D63/02)}
- C12M 41/14: {Incubators; Climatic chambers (per se B01L1/00)}
- C12M 41/34: {of gas}
- C12M 41/44: {of volume or liquid level}
- C12M 41/48: {Automatic or computerized control (automatic analysis G01N35/00)}
- C12N 5/00: Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00)
- C12N 5/0062: {General methods for three-dimensional culture}
- C12N 5/0658: {Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts}
- 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}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Proposed are a three-dimensional structuring method of cells and a three-dimensional structuring system of cells capable of efficiently bonding multiple cell clusters in a three-dimensional direction, pursuant to their growth, while ensuring safety. A plurality of fibers, in which one end of each of the fibers is held by a flat plate, are inserted together with the flat plate into a flow path through which a culture solution is supplied, a plurality of cell clusters are placed in the flow path upon causing the cell clusters to run with a liquid flow of the culture solution, and each of the cell clusters is cultured by being stacked on an outer surface of each of the fibers with the flat plate as a growth origin.

## Claims

### Claim 1

A three-dimensional culture method of cells, comprising the steps of:
positioning a plurality of fibers in a flow path spatial part, in which a space surrounded by an inner wall surface from a supply port to a discharge port of a culture solution is used as a flow path, so that a longitudinal direction of each of the fibers is along a direction of the flow path while a predetermined interval is mutually maintained between the fibers; and
placing a plurality of cell clusters in the flow path upon causing the cell clusters to run with a liquid flow of the culture solution supplied in the flow path from the supply port, and culturing each of the cell clusters by stacking the cell clusters on an outer surface of each of the fibers with a vicinity of the discharge port as a growth origin.

### Claim 2 (depends on 1)

The three-dimensional culture method of cells according to claim 1,
wherein each of the fibers has a hollow or a lateral groove along a longitudinal direction formed therein, has a plurality of micropores that penetrate from the outer surface to the hollow interior or the lateral groove interior, and metabolic waste is removed from each of the cell clusters through each of the micropores.

### Claim 3 (depends on 2)

The three-dimensional culture method of cells according to claim 2,
wherein each of the fibers supplies either or both of growth factors and nutritional factors to each of the cell clusters through each of the micropores.

### Claim 4 (depends on 1)

The three-dimensional culture method of cells according to claim 1,
wherein a flow rate of the culture solution is controlled, and a number of the cell clusters to be placed in the flow path and a placement timing thereof are adjusted in accordance with a control state of the culture solution.

### Claim 5 (depends on 1)

The three-dimensional culture method of cells according to claim 1,
wherein at least one or more among a temperature, a carbon dioxide concentration, an oxygen concentration and a nitrogen concentration of the culture solution are controlled.

### Claim 6 (depends on 1)

The three-dimensional culture method of cells according to claim 1,
wherein subtle vibration is applied to each of the fibers, along a flow path direction, at a predetermined timing in accordance with a growth process of each of the cell clusters.

### Claim 7

A three-dimensional culture system of cells, comprising:
a flow path spatial part in which a space surrounded by an inner wall surface from a supply port to a discharge port of a culture solution is used as a flow path;
a plurality of fibers which are positioned so that a longitudinal direction of each of the fibers is along a direction of the flow path while a predetermined interval is mutually maintained between the fibers; and
a liquid supplying part which supplies the culture solution in the flow path from the supply port of the flow path spatial part,
wherein a plurality of cell clusters are placed in the flow path upon causing the cell clusters to run with a liquid flow of the culture solution supplied in the flow path from the supply port, and each of the cell clusters is cultured by stacking the cell clusters on an outer surface of each of the fibers with a vicinity of the discharge port as a growth origin.

### Claim 8 (depends on 7)

The three-dimensional culture system of cells according to claim 7,
wherein the flow path spatial part is a closed space formed within a ring of an annular seal member by bonding a first case and a second case, in which grooves of a predetermined shape are formed on either or both of opposing surfaces, by interposing the seal member so as to surround the grooves.

### Claim 9 (depends on 8)

The three-dimensional culture system of cells according to claim 8,
wherein, with each of the fibers, the supply port side is blocked and the discharge port side is opened in the flow path spatial part.

### Claim 10 (depends on 8)

The three-dimensional culture system of cells according to claim 8,
wherein each of the fibers is supported in the flow path spatial part so that tensile force is applied between the blocked side and the open side.

### Claim 11 (depends on 7)

The three-dimensional culture system of cells according to claim 7,
wherein each of the fibers has a hollow or a lateral groove along a longitudinal direction formed therein, has a plurality of micropores that penetrate from the outer surface to the hollow interior or the lateral groove interior, and metabolic waste is removed from each of the cell clusters through each of the micropores.

### Claim 12 (depends on 7)

The three-dimensional culture system of cells according to claim 7,
wherein each of the fibers supplies either or both of growth factors and nutritional factors to each of the cell clusters through each of the micropores.

### Claim 13 (depends on 7)

The three-dimensional culture system of cells according to claim 7, further comprising:
a control unit which controls a flow rate of the culture solution.

### Claim 14 (depends on 13)

The three-dimensional culture system according to claim 13,
wherein the control unit controls at least one or more among a temperature, a carbon dioxide concentration, an oxygen concentration and a nitrogen concentration of the culture solution.

### Claim 15 (depends on 13)

The three-dimensional culture system according to claim 13, further comprising:
a cell placement adjustment unit which adjusts a number of the cell clusters to be placed in the flow path and a placement timing thereof in accordance with a control state of the culture solution by the control unit.

### Claim 16 (depends on 13)

The three-dimensional culture system of cells according to claim 13, further comprising:
a vibration application part which alternately applies subtle vibration to each of the fibers in a flow path direction or a reverse direction in accordance with a growth process of each of the cell clusters under control of the control unit.