# US 9,518,249 B2 · Cell carrier, associated methods for making cell carrier and culturing cells using the same

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- Number: US 9,518,249 B2
- Document ID: US9518249B2
- Status: Granted
- Kind: B2
- Application number: 13/839,049
- Filed: 2013-03-15
- Published: 2014-07-03
- Granted: 2016-12-13
- Earliest claimed: 2010-12-16
- Assignees: General Electric Company
- Applicants: General Electric Company
- Inventors: Brian Michael Davis; Evelina Roxana Loghin; Kenneth Roger Conway; Andrew Arthur Paul Burns; David Gilles Gascoyne
- Web page: https://patentgrep.com/patent/US9518249B2

## CPC

- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- B29C 37/0025: {Applying surface layers, e.g. coatings, decorative layers, printed layers, to articles during shaping, e.g. in-mould printing (moulding on preformed layers as inserts B29C70/68; applying fluent material to surfaces in general B05)}
- B29C 59/02: by mechanical means, e.g. pressing {(B29C59/007 takes precedence; embossing expanded porous articles B29C44/5627)}
- B29C 71/0081: {using an electric field, e.g. for electrostatic charging (electrostatic pinning of extruded material B29C48/9165; fixing linings by electrostatic charges B29C63/0043)}
- C12M 23/12: {Well or multiwell plates (C12M25/04 takes precedence)}
- C12M 23/20: {Material Coatings (immunocoatings C12M25/00)}
- B29K 2025/06: {PS, i.e. polystyrene}
- B29K 2105/256: {Sheets, plates, blanks or films}
- B29K 2883/00: Use of polymers having silicon, with or without sulfur, nitrogen, oxygen, or carbon only, in the main chain, as mould material
- B29L 2031/712: {Containers; Packaging elements or accessories, Packages (closures therefor B29L2031/56; ink or toner cartridges B29L2031/7678; squeeze tubes B29L2023/20; suitcases B29L2031/7418)}
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2533/30: Synthetic polymers
- C12N 2533/52: Fibronectin; Laminin
- C12N 2533/54: Collagen; Gelatin
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- C12N 2535/10: Patterned coating
- C12N 2537/00: Supports and/or coatings for cell culture characterised by physical or chemical treatment
- C12N 2539/10: Coating allowing for selective detachment of cells, e.g. thermoreactive coating
- Y10T 156/1039: Surface deformation only of sandwich or lamina [e.g., embossed panels]
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- B29C: SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING (making preforms B29B11/00; making laminated products by combining previously unconnected layers which become one product whose layers will remain together B32B37/00 - B32B41/00)
- 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}
- B29K: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES B29B, B29C OR B29D, RELATING TO MOULDING MATERIALS OR TO MATERIALS FOR {MOULDS, } REINFORCEMENTS, FILLERS OR PREFORMED PARTS, e.g. INSERTS
- B29L: INDEXING SCHEME ASSOCIATED WITH SUBCLASS B29C, RELATING TO PARTICULAR ARTICLES
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

A carrier for expansion of pluripotent stem cells is provided, wherein the carrier comprises a substrate comprising one or more outer surfaces, wherein the one or more outer surfaces are modified with gas plasma treatment, and one or more structured indentations on one or more of the outer surfaces. The carrier has a length at least about 0.2 mm, a width at least about 0.2 mm, and a height in a range from about 0.05 mm to 1.2 mm and each of the structured indentations has a major axis in a range from about 0.1 mm to 0.5 mm, a minor axis in a range from about 0.1 mm to 0.5 mm and a depth in a range from about 0.025 mm to about 0.5 mm. A method of making the carrier, and culturing stromal cells using the same carrier are also provided.

## Claims

### Claim 1

A carrier for expansion of pluripotent stem cells, comprising:
a planar substrate disc comprising a first surface and a second surface opposing the first surface and a plurality of structured indentations formed in the first surface and the second surface;
wherein the first surface and the second surface are modified with gas plasma treatment, and
wherein the planar substrate disc has a length at least about 0.2 mm, a width at least about 0.2 mm, and a height in a range from about 0.05 mm to 1.2 mm, wherein the height extends from a highest point of the first surface through the planar substrate disc and to a highest point of the second surface and each of the structured indentations has a major axis in a range from about 0.1 mm to 0.5 mm, a minor axis in a range from about 0.1 mm to 0.5 mm and a depth in a range from about 0.025 mm to 0.5 mm and wherein a first structured indentation of the plurality of structured indentations is aligned with a second structured indentation of the plurality of structured indentations in the planar substrate disc such that respective bottoms of the first structured indentation and the second structured indentation are separated by a portion of the planar substrate disc.

### Claim 2 (depends on 1)

The carrier of claim 1, wherein the first surface and the second surface are configured for expansion of pluripotent stem cells.

### Claim 3 (depends on 1)

The carrier of claim 1, wherein the gas plasma treatment comprises plasma treatment with one or more gases comprising oxygen, nitrogen, amonia, carbon dioxide or combinations thereof.

### Claim 4 (depends on 1)

The carrier of claim 1, wherein the gas plasma treatment comprises oxygen plasma treatment.

### Claim 5 (depends on 1)

The carrier of claim 1, further comprising a biomolecular coating disposed on one or more of the first surface or the second surface.

### Claim 6 (depends on 5)

The carrier of claim 5, wherein the biomolecular coating comprises proteins or peptides.

### Claim 7 (depends on 5)

The carrier of claim 5, wherein the biomolecular coating comprises proteins.

### Claim 8 (depends on 7)

The carrier of claim 7, wherein the proteins comprise collagen, vitronectin, fibronectin, laminin, recombinant collagen, recombinant vitronectin, recombinant e-cadherin or combinations thereof.

### Claim 9 (depends on 5)

The carrier of claim 5, wherein the biomolecular coating comprises synthetic peptides.

### Claim 10 (depends on 5)

The carrier of claim 5, wherein the biomolecular coating comprises extracellular matrix (ECM) proteins, proteoglycans, factors derived from a mouse sarcoma cell line, or combinations thereof.

### Claim 11 (depends on 5)

The carrier of claim 5, wherein the biomolecular coating forms a cytophilic surface.

### Claim 12 (depends on 1)

The carrier of claim 1, wherein each of the structured indentations comprises a cross sectional profile of polygonal, circular, or elliptical shape.

### Claim 13 (depends on 1)

The carrier of claim 1, wherein each of the structured indentations has an aspect ratio in a range from about 0.1 to about 1.5.

### Claim 14 (depends on 1)

The carrier of claim 1, wherein the carrier is made of glass, a polymer, a ceramic, a metal, or combinations thereof.

### Claim 15 (depends on 1)

The carrier of claim 1, wherein the carrier is made of dextran, silicone, polyester, polycarbonate, polyamide, polyurethane, olefin polymer, or polyacrylate polymer.

### Claim 16 (depends on 1)

The carrier of claim 1, wherein the carrier is made of polystyrene.

### Claim 17 (depends on 1)

The carrier of claim 1, having a perimeter that is triangular, rectangular, square, pentagonal, hexagonal, circular, or elliptical.

### Claim 18 (depends on 1)

A cell culture system comprising at least one of the carrier of claim 1.

### Claim 19 (depends on 18)

The cell culture system of claim 18, wherein the cell culture system is a bioreactor.

### Claim 20 (depends on 19)

The cell culture system of claim 19, wherein the bioreactor comprises a fluid having a forced convective fluid motion.

### Claim 21 (depends on 20)

The cell culture system of claim 20, wherein the bioreactor is a stirred tank bioreactor, or a reactor with rocking or rolling motion.

### Claim 22 (depends on 1)

A kit for culturing cells, comprising a disposable housing pre-loaded with the carrier of claim 1.

### Claim 23 (depends on 22)

The kit of claim 22, wherein the disposable housing comprises a bag, a flask, a tube, a petridish, or a bottle.

### Claim 24

A carrier for expansion of pluripotent stem cells, comprising:
a planar substrate comprising a first surface and a second surface opposing the first surface and a plurality of structured indentations on the first surface and the second surface;
wherein the first surface and the second surface comprise a biomolecular coating,
wherein the planar substrate has a length at least about 0.2 mm, a width at least about 0.2 mm, and a height in a range from about 0.05 mm to 1.2 mm, wherein the height extends from a highest point of the first surface through the planar substrate and to a highest point of the second surface, and wherein each of the structured indentations has a major axis in a range from about 0.1 mm to 0.5 mm, a minor axis in a range from about 0.1 mm to 0.5 mm and a depth in a range from about 0.025 mm to 0.5 mm and wherein a first structured indentation of the plurality of structured indentations is aligned with a second structured indentation of the plurality of structured indentations in the planar substrate such that respective bottoms of the first structured indentation and the second structured indentation are separated by a portion of the planar substrate; and
a plurality of pluripotent stem cells disposed in a least one of the plurality of structured indentations.

### Claim 25 (depends on 24)

The carrier of claim 24, wherein the biomolecular coating comprises proteins or peptides.

### Claim 26

A carrier for expansion of pluripotent stem cells, comprising:
a planar substrate comprising a first surface and a second surface opposing the first surface modified with one or more of corona discharge treatment, gas plasma treatment, or chemical functionalization to form modified surfaces and a plurality of structured indentations on the first surface and the second surface;
a biomolecular coating disposed on the first or second surface,
wherein the carrier has a length at least about 0.2 mm, a width at least about 0.2 mm, and a height in a range from about 0.05 mm to 1.2 mm wherein the height extends from a highest point of the first surface through the planar substrate and to a highest point of the second surface, and each of the structured indentations has a major axis in a range from about 0.1 mm to 0.5 mm, a minor axis in a range from about 0.1 mm to 0.5 mm, and a depth in a range from about 0.025 mm to about 0.5 mm and wherein a first structured indentation of the plurality of structured indentations is aligned with a second structured indentation of the plurality of structured indentations in the planar substrate such that respective bottoms of the first structured indentation and the second structured indentation are separated by a portion of the planar substrate; and
a plurality of pluripotent stem cells disposed in at least one of the plurality of indentations.

### Claim 27 (depends on 26)

The carrier of claim 26, wherein the gas plasma treatment comprises plasma treatment with one or more of the gases comprising oxygen, nitrogen, amonia, carbon dioxide, or combinations thereof.