# US 10,870,824 B2 · Cell support composite and method for producing cell support composite

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- Number: US 10,870,824 B2
- Document ID: US10870824B2
- Status: Granted
- Kind: B2
- Application number: 15/970,092
- Filed: 2018-05-03
- Published: 2018-08-30
- Granted: 2020-12-22
- Earliest claimed: 2015-11-10
- Assignees: Nikkiso Co., Ltd.
- Applicants: Nikkiso Co., Ltd.
- Inventors: Fumihiko Kitagawa
- Web page: https://patentgrep.com/patent/US10870824B2

## CPC

- C12M 25/06: {Plates; Walls; Drawers; Multilayer plates}
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- C12M 25/00: {Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings}
- C12M 25/10: {Hollow fibers or tubes (hollow fiber modules in general B01D63/02)}
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12N 5/0686: {Kidney cells}
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C12N 2533/52: Fibronectin; Laminin
- 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}
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A cell support composite includes: a substrate formed of an artificial material; a laminin molecule or a fragment thereof which adheres to at least a part of the substrate; and a renal tubule epithelial cell or a renal tubule epithelial-like cell which is a culture cell attached to the substrate via the laminin molecule or the fragment thereof.

## Claims

### Claim 1

A cell support composite comprising:
a substrate formed of an artificial material;
a laminin molecule or a fragment thereof which adheres to at least a part of the substrate; and
a monolayer of a renal tubule epithelial cell or a renal tubule epithelial-like cell which is a cultured cell attached to the substrate via the laminin molecule or the fragment thereof, wherein the monolayer of the renal tubule epithelial cell or the renal tubule epithelial-like cell is maintained for 15 days or more,
wherein the laminin molecule or the fragment thereof is selected from any one of
Laminin-111 or a fragment thereof that adheres to the substrate at an amount of 0.15 μg/cm2 or more,
Laminin-211 or a fragment thereof that adheres to the substrate at an amount of 0.52 μg/cm2 or more,
Laminin-221 or a fragment thereof that adheres to the substrate at an amount of 0.34 μg/cm2,
Laminin-311 or a fragment thereof that adheres to the substrate at a maximum amount after air drying of 0.5 μg/cm2 or more,
Laminin-332 or a fragment thereof that adheres to the substrate at a maximum amount after air drying of 1.2 μg/cm2 or more,
Laminin-421 or a fragment thereof that adheres to the substrate at an amount of 0.50 μg/cm2 or more,
Laminin-511 or a fragment thereof that adheres to the substrate at an amount of 0.32 μg/cm2 or more,
a variant of E8 region of Laminin-511 that adheres to the substrate at an amount of 0.15 μg/cm2 or more, and
Laminin-521 or a fragment thereof that adheres to the substrate at an amount of 0.44 μg/cm2 or more.

### Claim 2 (depends on 1)

The cell support composite according to claim 1, wherein the laminin 311 or the fragment thereof adheres to the substrate at an amount of 0.15 μg/cm2 or more.

### Claim 3 (depends on 1)

The cell support composite according to claim 1,
wherein the cultured cell has formed a confluent single layer without being substantially multilayered.

### Claim 4 (depends on 1)

A method for producing the cell support composite of claim 1, comprising:
coating at least a part of a substrate formed of an artificial material with a laminin molecule or a fragment thereof;
seeding a renal tubule epithelial cell or a renal tubule epithelial-like cell which is a cultured cell on the laminin molecule or the fragment thereof that has adhered to the substrate; and
culturing the cultured cell to give a single layer structure of the cultured cell,
wherein the coating with the laminin molecule or the fragment thereof comprises any one of:
coating with Laminin-111 at a concentration of 3.0 mg/ml or more to give an adhesion amount of 0.15 mg/cm2 or more;
coating with Laminin-211 at a concentration of 10.0 mg/ml or more to give an adhesion amount of 0.52 mg/cm2 or more;
coating with Laminin-221 at a concentration of 10.0 mg/ml or more to give an adhesion amount of 0.34 mg/cm2 or more;
coating with Laminin-311 at a concentration of 3.0 mg/ml or more to give a maximum adhesion amount after air drying of 0.5 mg/cm2 or more;
coating with Laminin-332 at a concentration of 5.0 mg/ml or more to give a maximum adhesion amount after air drying of 1.2 mg/cm2 or more;
coating with Laminin-421 at a concentration of 5.0 mg/ml or more to give an adhesion amount of 0.50 mg/cm2 or more;
coating with Laminin-511 at a concentration of 10.0 mg/ml or more to give an adhesion amount of 0.32 mg/cm2 or more;
coating with a variant of E8 region of Laminin-511 at a concentration of 1.4 mg/ml or more to give an adhesion amount of 0.15 mg/cm2 or more; and
coating with Laminin-521 at a concentration of 5.0 mg/ml or more to give an adhesion amount of 0.44 mg/cm2 or more.

### Claim 5 (depends on 4)

The method for producing a cell support composite according to claim 4, wherein the coating with the laminin molecule or the fragment thereof comprises any one of:
coating with Laminin-311 at a concentration of 4.0 mg/ml or more to give an adhesion amount of 0.15 mg/cm2 or more;
coating with Laminin-511 at a concentration of 10.0 mg/ml or more to give an adhesion amount of 0.32 mg/cm2 or more;
coating with a variant of E8 region of Laminin-511 at a concentration of 1.4 mg/ml or more to give an adhesion amount of 0.15 mg/cm2 or more; and
coating with Laminin-521 at a concentration of 5.0 mg/ml or more to give an adhesion amount of 0.44 mg/cm2 or more.

### Claim 6 (depends on 4)

The method for producing a cell support composite according to claim 4, wherein the cultured cell has formed a confluent single layer without being substantially multilayered.