# US 10,479,974 B2 · Method, device and kit for mass cultivation of cells using polyimide porous membrane

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- Number: US 10,479,974 B2
- Document ID: US10479974B2
- Status: Granted
- Kind: B2
- Application number: 15/545,609
- Filed: 2016-01-26
- Published: 2018-01-18
- Granted: 2019-11-19
- Earliest claimed: 2015-01-26
- Assignees: Ube Industries, Ltd.
- Applicants: Ube Industries, Ltd.
- Inventors: Masahiko Hagihara; Motohisa Shimizu; Yukinori Wada
- Web page: https://patentgrep.com/patent/US10479974B2

## CPC

- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- A01N 1/128: Chemically defined matrices for immobilising, holding or storing living parts, e.g. alginate gels; Chemically altering living parts, e.g. by cross-linking
- C07K 14/535: Granulocyte CSF; Granulocyte-macrophage CSF
- C12M 23/20: {Material Coatings (immunocoatings C12M25/00)}
- C12M 25/02: {Membranes; Filters (filters or filtration in general B01D24/00-B01D41/00)}
- C12M 27/16: {Vibrating; Shaking; Tilting}
- C12M 29/00: {Means for introduction, extraction or recirculation of materials, e.g. pumps (pumps per se F04B)}
- C12N 1/14: Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor
- C12N 1/16: Yeasts; Culture media therefor
- C12N 1/20: Bacteria; Culture media therefor
- C12N 5/04: Plant cells or tissues {(culture media C12N5/0025)}
- C12N 5/0656: {Adult fibroblasts}
- C12N 5/0682: {Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells (oocytes C12N5/0609)}
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12N 2533/30: Synthetic polymers
- C12N 2535/00: Supports or coatings for cell culture characterised by topography
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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}
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The present invention pertains to a method for the mass cultivation of cells, and a cell cultivation device and kit. The present invention further pertains to a continuous cell cultivation method and a continuous cell cultivation device in which a carrier is used.

## Claims

### Claim 1

A mass cell culturing method including:
(1) applying cells to a porous polyimide film, and
(2) applying the porous polyimide film to which the cells have been applied, to a cell culture medium and performing culturing the porous polyimide film to which the cells have been applied, until the number of cells reaches 1.0×106 or more per milliliter of medium, or until the number of cells per square centimeter of the porous polyimide film reaches 1.0×105,
wherein the porous polyimide film has a multilayer structure having at least two surface layers (an A-surface and a B-surface), and a macro-void layer sandwiched between the two surface layers,
a mean pore size of the holes in the A-surface is smaller than a mean pore size of the holes in the B-surface, and
the macro-void layer has a partition bonded to the surface layers (the A-surface and the B-surface), and a plurality of macro-voids surrounded by the partition and the surface layers (the A-surface and the B-surface).

### Claim 2 (depends on 1)

The method according to claim 1, wherein two or more porous polyimide films layered either above and below or left and right are used in the cell culture medium.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the porous polyimide films are:
i) folded,
ii) wound into a roll,
iii) connected as sheets or fragments by a filamentous structure, or
iv) bound into a rope,
to be suspended or fixed in the cell culture medium in the cell culturing vessel.

### Claim 4 (depends on 1)

The method according to claim 1, wherein in the culturing of step (2), all or some of the porous polyimide films are not in contact with the liquid phase of the cell culture medium.

### Claim 5 (depends on 1)

The method according to claim 1, wherein in the culturing of step (2), the total volume of the cell culture medium in the cell culturing vessel is 10,000 times or less of the total sum of the porous polyimide film volume including the cell survival zone.

### Claim 6 (depends on 1)

The method according to claim 1, wherein in the culturing of step (2), the total volume of the cell culture medium in the cell culturing vessel is 100 times or less of the total sum of the porous polyimide film volume including the cell survival zone.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the culturing in step (2) is carried out in a system in which a cell culture medium is continuously or intermittently supplied to a cell culturing vessel from cell culture medium supply means installed outside of the cell culturing vessel.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the cell culture medium is circulated between the cell culture medium supply means and the cell culturing vessel.

### Claim 9 (depends on 7)

The method according to claim 7, wherein the system is a cell culturing apparatus including a culturing unit which is the cell culturing vessel, and a culture medium-supply unit which is the cell culture medium supply means, wherein
the culturing unit is a culturing unit that houses one or more porous polyimide films to support cells, and that comprises a culture medium supply port and a culture medium discharge port, and
the culture medium-supply unit is a culture medium-supply unit comprising a culture medium housing vessel, a culture medium supply line, and a liquid conveyance pump that conveys the medium continuously or intermittently through the culture medium supply line, the first end of the culture medium supply line contacting the medium in the culture medium housing vessel, and the second end of the culture medium supply line being in communication with the culturing unit interior via the culture medium supply port of the culturing unit.

### Claim 10 (depends on 9)

The method according to claim 9, wherein the culturing unit is a culturing unit that does not comprise an air supply port and air discharge port.

### Claim 11 (depends on 9)

The method according to claim 9, wherein the culturing unit further comprises a culture medium discharge line, the first end of the culture medium discharge line being connected to the culture medium housing vessel, the second end of the culture medium discharge line being in communication with the culturing unit interior via the culture medium discharge port of the culturing unit, and the medium being able to circulate through the culture medium-supply unit and the culturing unit.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the cells are selected from the group consisting of animal cells, insect cells, plant cells, yeast cells and bacteria.

### Claim 13 (depends on 12)

The method according to claim 12, wherein the animal cells are cells derived from an animal belonging to the subphylum Vertebrata.

### Claim 14 (depends on 12)

The method according to claim 12, wherein the bacteria are selected from the group consisting of lactic acid bacteria, E. coli, Bacillus subtilis and cyanobacteria.

### Claim 15 (depends on 1)

The method according to claim 1, wherein the porous polyimide film is a porous polyimide film including a polyimide obtained from a tetracarboxylic dianhydride and a diamine.

### Claim 16 (depends on 15)

The method according to claim 15, wherein the porous polyimide film is a colored porous polyimide film obtained by forming a polyamic acid solution composition including a polyamic acid solution obtained from a tetracarboxylic dianhydride and a diamine, and a coloring precursor, and then heat treating it at 250° C. or higher.