# US 5,700,681 A · Selection of cells having increased cell adhesion properties

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- Number: US 5,700,681 A
- Document ID: US5700681A
- Status: Granted
- Kind: A
- Application number: 08/359,956
- Filed: 1994-12-20
- Granted: 1997-12-23
- Earliest claimed: 1987-09-28
- Assignees: La Jolla Cancer Research Foundation
- Inventors: Michael D. Pierschbacher; Erkki I. Ruoslahti; Shoukat Dedhar
- Web page: https://patentgrep.com/patent/US5700681A

## CPC

- C12N 5/0693: {Tumour cells; Cancer cells}
- C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)}
- C12N 2501/58: Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
- C12N 2501/998: Proteins not provided for elsewhere
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method for selecting cell lines expressing increased cell adhesion properties or promoting cell differentiation by culturing cells in increasing concentrations of adhesion ligand containing media. Cell lines produced by such method.

## Claims

### Claim 1

A method for promoting differentiation of animal cells and preventing a differentiated state of animal cells from defifferentiating, comprising the steps of:
a. providing an initial sample of said animal cells;
b. culturing said animal cells in culture media containing an initial concentration of an RGD containing adhesion ligand in solution,
wherein said RGD-containing adhesion ligand binds to RGD binding receptors on the surface of said animal cells which inhibits the adhesive function of said receptors, and wherein said initial concentration is selected so that not every receptor is bound;
c. choosing those cells which proliferate in the presence of said initial concentration of said RGD containing adhesion ligand in solution;
d. successively reculturing the chosen cells in culture media containing successively increasing concentrations of said adhesion ligand in solution;
e. selecting those cells which proliferate in concentrations of said adhesion ligand in solution normally inhibitory to cell proliferation; and
f. establishing from the selected cells a cell line which is more differentiated than said initial sample of animal cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein said RGD-containing adhesion ligand in solution is a peptide.

### Claim 3 (depends on 2)

The method of claim 2, wherein said peptide is GRGDSP.

### Claim 4 (depends on 1)

The method of claim 1, further comprising a step of coating the substrate of a vessel in which said initial sample of animal cells are cultured with an adhesion promoting material which binds to the same cell surface receptors as said RGD-containing adhesion ligand in solution.

### Claim 5 (depends on 1)

The method of claim 1, further comprising:
adding to the animal cell culture, to which said RGD containing adhesion ligand has been added, a receptor blocking molecule,
wherein said receptor blocking molecule binds to cell surface receptors which do not bind said RGD-containing adhesion ligand.

### Claim 6 (depends on 5)

The method of claim 5, wherein said receptor blocking molecule is provided in sufficient concentration to bind to about all of its corresponding cell surface receptor.

### Claim 7 (depends on 5)

The method of claim 5 wherein said receptor blocking molecule will bind to fibronectin receptors.

### Claim 8 (depends on 1)

The method of claim 1, wherein said initial sample of animal cells is selected from the group consisting of tumor cells, undifferentiated malignant cells, normal undifferentiated stem cells and dedifferentiated cells.

### Claim 9 (depends on 1)

The method of claim 1, wherein said initial sample of animal cells are osteosarcoma cells.

### Claim 10 (depends on 1)

The method of claim 1, wherein said initial sample of animal cells are MG-63 cells.

### Claim 11 (depends on 1)

The method of claim 1, wherein said initial sample of animal cells are non-malignant cells.

### Claim 12

A method for promoting the differentiation of animal cells and preventing a differentiated state of animal cells from dedifferentiating comprising the steps of:
a. providing an initial sample of said animal cells;
b. culturing said animal cells in culture media containing an initial concentration of an antibody in solution,
wherein said antibody binds to RGD binding receptors on the surface of said animal cells which inhibits the adhesive function of said RGD binding receptors, and wherein said initial concentration is selected so that not every RGD binding receptor is bound;
c. choosing those cells which proliferate in the presence of said initial concentration of said antibody in solution;
d. successively reculturing said chosen cells in culture media containing successively increasing concentrations of said antibody in solution;
e. selecting those cells which proliferate in concentrations of said antibody in solution normally inhibitory to cell proliferation; and
f. establishing from said selected cells a cell line which is more differentiated than said initial sample of animal cells.

### Claim 13 (depends on 1, 12)

The method of claim 1 or 12, wherein said cell line of step (f) contains cells that do not proliferate or proliferate at a slower rate than animal cells in a less differentiated state.

### Claim 14

A method of increasing the differentiated state of undifferentiated malignant animal cells, normal undifferentiated animal stem cells, and dedifferentiated animal cells, comprising the steps of:
a) reacting a soluble RGD-containing adhesion ligand at an initial concentration with a sample of animal cells selected from the group consisting of undifferentiated malignant animal cells, normal undifferentiated animal stem cells, and dedifferentiated animal cells;
b) permitting said RGD-containing adhesion ligand to specifically bind to RGD binding cell surface receptors on the animal cells;
c) identifying the subset of animal cells which have bound the highest concentration of said RGD-containing adhesion ligand to their RGD binding cell surface receptors;
d) isolating culturing said subset of animal cells in the presence of a concentration of said RGD-containing adhesion ligand that is higher than the concentration in step (a);
e) collecting the animal cells from step (d) and reacting them with said RGD-containing adhesion ligand at a concentration equal to the concentration of RGD-containing adhesion ligand in step (d); and
f) repeating steps (b) through (e) until the differentiated state of the animal cells isolated in step (d) is increased.

### Claim 15

A method for promoting the differentiation of animal cells and maintaining a differentiated state of animal cells likely to dedifferentiate, comprising the steps of:
a) providing a soluble RGD-remaining adhesion ligand at an initial concentration wherein said RGD-containing ligand binds selectively to RGD binding cell surface receptors expressed by animal cells;
b) permitting said RGD-containing ligand to react with a sample of animal cells;
c) identifying the animal cells which have bound the highest concentration of said RGD-containing adhesion ligand to their RGD binding cell surface receptors;
d) isolating and culturing said animal cells identified in step (c) in the presence of a concentration of said RGD-containing adhesion ligand that is higher than the concentration in step (a);
e) collecting the animal cells from the culture of step (d) and reacting them with said RGD-containing adhesion ligand at a concentration equal to the concentration of RGD-containing adhesion ligand in step (d); and
f) repeating steps (b) through (e) until the desired differentiated state of the animal cells isolated in step (d) is obtained.

### Claim 16 (depends on 14, 15)

The method of claim 14 or 15, wherein said animal cells of step (f) do not proliferate or proliferate at a slower rate than animal cells in a less differentiated state.

### Claim 17 (depends on 1, 12, 14, 15)

The method of claim 1, 12, 14 or 15, wherein said animal cells are insulin-secreting pancreatic cells.

### Claim 18 (depends on 1, 12, 14, 15)

The method of claim 1, 12, 14 or 15, wherein said animal cells are bone producing cells.

### Claim 19 (depends on 1, 12, 14, 15)

The method of claim 1, 12, 14 or 15, wherein said animal cells in a differentiated state have increased adhesive properties.

### Claim 20 (depends on 14, 15)

The method of claims 14 or 15, further comprising:
establishing from animal cells in step (f) a cell line which is more differentiated than said sample of animal cells.