# US 5,702,949 A · Culture method for multilayer growth of anchorage-dependent cells

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- Number: US 5,702,949 A
- Document ID: US5702949A
- Status: Granted
- Kind: A
- Application number: 08/665,822
- Filed: 1996-06-19
- Granted: 1997-12-30
- Earliest claimed: 1996-06-19
- Assignees: University of Utah Research Foundation
- Inventors: Edward M. Trujillo; Catherine Rappaport
- Web page: https://patentgrep.com/patent/US5702949A

## CPC

- C12M 23/20: {Material Coatings (immunocoatings C12M25/00)}
- C12M 25/06: {Plates; Walls; Drawers; Multilayer plates}
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12N 2533/20: Small organic molecules
- C12N 2533/54: Collagen; Gelatin
- 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

Anchorage-dependent cells are grown on a novel substratum which is believed to increase the oxygenation of the cells and reduce the formation of free-radicals. The substratum consists of a perfluorocarbon reservoir which is coated with a layer of protein (e.g.,collagen or gelatin) which has been perfluoroalkylated. The perfluoroalkylated protein forms a very stable surface to which anchorage-dependent cells can attach and grow. Using this system, mammalian cell cultures have been grown to densities exceeding 10.sup.7 cells/cm.sup.2. The increased density is attributed to the formation of multiple cell layers which resemble tissue-like structures.

## Claims

### Claim 1

A method for the attachment and growth of cells comprising the steps of:
a. contacting a surface with a perfluorocarbon;
b. bonding a perfluoroalkylated protein to the perfluorocarbon such that a perfluoroalkylated/perfluorocarbon substratum is formed;
c. adding aqueous growth media; and
d. contacting the perfluoroalkylated/perfluorocarbon substratum with cells.

### Claim 2 (depends on 1)

The method of attaching and growing cells of claim 1 wherein the perfluorocarbon is selected from the group consisting of perfluorodecalin, perfluorotributylamine and perfluorobutyltetrahydrofuran.

### Claim 3 (depends on 1)

The method of attaching and growing cells of claim 1 wherein the perfluoroalkylated protein has a perfluorocarbon tail, and wherein the perfluorocarbon tail is perfluorooctylpropyl.

### Claim 4 (depends on 1)

The method of attaching and growing cells of claim 1 wherein the perfluoroalkylated protein is selected from the group consisting of gelatins, collagens, fibronectins, laminins and elastins.

### Claim 5 (depends on 1)

The method of attaching and growing cells of claim 1 wherein the cells are eukaryotic cells.

### Claim 6 (depends on 5)

The method of attaching and growing cells of claim 5 wherein the cells are co-cultured.

### Claim 7 (depends on 1)

The method of attaching and growing cells of claim 1 wherein the eukaryotic cells are selected from the group consisting of HeLa cells, hepatocytes, liver cells, cancer cells, bone marrow cells, nerve cells, heart cells and co-cultures of the above.

### Claim 8 (depends on 6)

The method of attaching and growing cells of claim 6 wherein the cells grow to form multiple cell layers following contact with the perfluoroalkylated/perfluorocarbon substratum.

### Claim 9 (depends on 1)

The method of attaching and growing cells of claim 1 wherein the aqueous growth media is not in direct contact with the perfluorocarbon.

### Claim 10 (depends on 1)

The method of attaching and growing cells of claim 1 wherein the surface contacted with perfluorocarbon is a closed system.

### Claim 11 (depends on 1)

The method of attaching and growing cells of claim 1 wherein the surface contacted with perfluorocarbon is an open system.

### Claim 12

A method for the attachment and growth of cells comprising the steps of:
a. contacting a surface with a perfluorocarbon;
b. perfluoroalkylating a protein;
c. bonding the perfluoroalkylated protein to the perfluorocarbon such that a perfluoroalkylated/perfluorocarbon substratum is formed;
d. contacting the substratum with aqueous growth media; and
e. contacting the perfluoroalkylated/perfluorocarbon substratum with at least one anchorage-dependent eukaryotic cells and allowing the anchorage-dependent eukaryotic cells to grow.

### Claim 13 (depends on 12)

The method of attaching and growing cells of claim 12 wherein the perfluorocarbon is selected from the group consisting of perfluorodecalin, perfluorotributylamine and perfluorobutyltetrahydrofuran.

### Claim 14 (depends on 12)

The method of attaching and growing cells of claim 12 wherein the protein is selected from the group consisting of gelatins, collagens, fibronectins, laminins and elastins.

### Claim 15 (depends on 12)

The method of attaching and growing cells of claim 12 wherein the eukaryotic cells are selected from the group consisting of HeLa cells, hepatocytes, liver cells, cancer cells, nerve cells, heart cells and co-cultures of the above.

### Claim 16 (depends on 12)

The method of attaching and growing cells of claim 12 wherein the cells form multiple cell layers.

### Claim 17 (depends on 12)

The method of attaching and growing cells of claim 12 wherein the aqueous growth media is not in direct contact with the perfluorocarbon.

### Claim 18 (depends on 12)

The method of attaching and growing cells of claim 12 wherein the surface contacted with perfluorocarbon is a closed system.

### Claim 19 (depends on 12)

The method of attaching and growing cells of claim 12 wherein the surface contacted with perfluorocarbon is an open system.