# US 10,479,971 B2 · Cell culture device and cell culture method

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- Number: US 10,479,971 B2
- Document ID: US10479971B2
- Status: Granted
- Kind: B2
- Application number: 15/649,652
- Filed: 2017-07-14
- Published: 2017-10-26
- Granted: 2019-11-19
- Earliest claimed: 2015-01-20
- Assignees: FUJIFILM Corporation
- Applicants: Fujifilm Corporation
- Inventors: Hideaki Kagawa; Shun Goto; Souichi Kohashi; Hidekazu Yamazaki
- Web page: https://patentgrep.com/patent/US10479971B2

## CPC

- C12M 21/08: {for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00)}
- C12M 27/02: {Stirrer or mobile mixing elements}
- C12M 29/00: {Means for introduction, extraction or recirculation of materials, e.g. pumps (pumps per se F04B)}
- C12M 33/14: {with filters, sieves or membranes}
- C12M 47/04: {Cell isolation or sorting (purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04)}
- C12M 47/20: {Heating or cooling (heating or cooling apparatus for laboratory uses B01L7/00)}
- C12N 1/00: Microorganisms; Compositions thereof (medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00); Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12M 33/12: {by pressure}
- 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

A culture container has a first inflow port and a first outflow port. The first flow path connects the first outflow port to the first inflow port. A storage container is provided within the first flow path and has a second inflow port which is connected to the first outflow port and a second outflow port which is connected to the first inflow port. A second flow path connects a first region within the first flow path, and a second region within the first flow path. A division processing portion is provided within the second flow path, performs a division process of dividing a cell aggregation flowing in from the first flow path, and allows the cells subjected to the division process to flow out into the first flow path via the second region. A medium supply portion supplies a medium to the inside of the first flow path.

## Claims

### Claim 1

A cell culture device comprising:
a culture container which has a first inflow port and a first outflow port and is used for culturing cells;
a first flow path which connects the first outflow port to the first inflow port;
a first storage container which is a storage container, which is provided within the first flow path and used for storing cells cultured in the culture container, the first storage container having a second inflow port which is connected to the first outflow port and a second outflow port which is connected to the first inflow port;
a second flow path connecting a first region within the first flow path which is positioned between the second outflow port and the first inflow port, and a second region within the first flow path which is positioned between the second inflow port and the first outflow port;
a division processing portion which is provided within the second flow path, performs a division process of dividing a cell aggregation, which is an aggregation of cells cultured in the culture container and flows in from the first flow path via the first region, and allows the cells subjected to the division process to flow out into the first flow path via the second region; and
a medium supply portion which supplies a medium to the inside of the first flow path.

### Claim 2 (depends on 1)

The cell culture device according to claim 1, further comprising:
a first stirring portion which is provided between the second outflow port and the first inflow port within the first flow path and stirs a fluid flowing in.

### Claim 3 (depends on 2)

The cell culture device according to claim 2,
wherein the first stirring portion includes a static mixer.

### Claim 4 (depends on 1)

The cell culture device according to claim 1, further comprising:
a first filter portion which is provided within a third flow path connecting the second inflow port to the second outflow port and performs a concentration process in which a liquid is removed from a mixture containing cells and the liquid accompanying the cells flowing out from the second outflow port.

### Claim 5 (depends on 4)

The cell culture device according to claim 4,
wherein the first filter portion includes a tangential flow filter.

### Claim 6 (depends on 4)

The cell culture device according to claim 4, further comprising:
a second storage container including a third outflow port and a third inflow port which is connected to the second inflow port; and
a second filter portion which is provided within a fourth flow path connecting the third inflow port to the third outflow port and performs a concentration process in which a liquid is removed from a mixture containing cells and the liquid accompanying the cells flowing out from the third outflow port.

### Claim 7 (depends on 6)

The cell culture device according to claim 6,
wherein the second filter portion includes a tangential flow filter.

### Claim 8 (depends on 6)

The cell culture device according to claim 6,
wherein the concentration process using the first filter portion and the concentration process using the second filter portion are alternately performed.

### Claim 9 (depends on 6)

The cell culture device according to claim 6, further comprising:
a first pressure adjustment mechanism, comprising a first air introducing valve and a first ventilation valve, which adjusts a pressure within the first storage container; and
a second pressure adjustment mechanism, comprising a second air introducing valve and a second ventilation valve, which adjusts a pressure within the second storage container.

### Claim 10 (depends on 1)

The cell culture device according to claim 1,
wherein the division processing portion has a processing container for performing the division process and a third pressure adjustment mechanism, comprising a third air introducing valve and a third ventilation valve, which adjusts a pressure within the processing container.

### Claim 11 (depends on 1)

The cell culture device according to claim 1, further comprising:
a second stirring portion which is provided within a fifth flow path connecting the medium supply portion and the first flow path and stirs a fluid flowing in.

### Claim 12 (depends on 11)

The cell culture device according to claim 11, further comprising:
a cell supply portion which is connected to a downstream side of the second stirring portion in the fifth flow path and supplies cells cultured in the culture container to the first flow path via the fifth flow path; and
a third stirring portion which is provided on a downstream side of a region, to which the cell supply portion is connected, in the fifth flow path and stirs a fluid flowing in.

### Claim 13 (depends on 1)

The cell culture device according to claim 1, further comprising:
a cell supply portion which supplies cells cultured in the culture container to the first flow path.

### Claim 14 (depends on 1)

The cell culture device according to claim 1, further comprising:
a freezing portion which is connected to a third region positioned between the second outflow port and the first outflow port, within the first flow path and freezes cells.

### Claim 15 (depends on 1)

The cell culture device according to claim 1, further comprising:
a pressure adjustment mechanism, comprising an air introducing valve and a ventilation valve, which adjusts a pressure within the culture container.

### Claim 16 (depends on 1)

A cell culture method in which the cell culture device according to claim 1 is used, the method comprising:
a medium exchange process of exchanging a used medium which has been used for culture of cells within the culture container with a new medium; and
a division process of dividing a cell aggregation formed through culturing cells in the culture container,
wherein the medium exchange process includes
a step of storing the cells cultured in the culture container in the first storage container together with a used medium,
a step of removing the used medium from a mixture containing the cells and the used medium which have been stored in the first storage container,
a step of supplying a new medium to the cells stored in the first storage container from the medium supply portion, and
a step of accommodating the mixture containing the cells and the new medium in the culture container, and
wherein the division process includes
a step of dividing the cell aggregation formed within the culture container in the division processing portion,
a step of exchanging a used medium which has been used for the culture of cells within the culture container with a new medium before or after dividing the cell aggregation in the division processing portion, and
a step of accommodating the mixture containing the divided cells and the new medium in the culture container.