# US 2014/0154735 A1 · Tumour cell and tissue culture

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- Number: US 2014/0154735 A1
- Document ID: US20140154735A1
- Status: Published application
- Kind: A1
- Application number: 13/978,209
- Filed: 2012-01-06
- Published: 2014-06-05
- Earliest claimed: 2011-01-06
- Assignees: Capsant Neurotechnologies S.A.
- Inventors: Lars Sundstrom; Thelma Biggs; Janet Foreman
- Web page: https://patentgrep.com/patent/US20140154735A1

## CPC

- G01N 33/5014: {for testing toxicity}
- C12N 5/0693: {Tumour cells; Cancer cells}
- G01N 33/5011: {for testing antineoplastic activity}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to in vitro three-dimensional organotypic cell co-culture. Cultures of the invention comprise tumour cells three-dimensionally disposed within a matrix of matrix cells which are distinct from the tumour cells, wherein the co-culture does not comprise a basement membrane. The cultures are useful for the study of cancers, for testing anti-tumour agent efficacy, and for high-throughput screening of candidate drugs.

## Claims

### Claim at position 1

-36. (canceled)

### Claim 37

An in vitro three-dimensional organotypic cell co-culture comprising tumour cells three-dimensionally disposed within a matrix of matrix cells which are distinct from the tumour cells, wherein the co-culture does not comprise a basement membrane.

### Claim 38 (depends on 37)

The co-culture of claim 37, wherein the co-culture further comprises a semi-permeable support and a liquid culture medium, wherein
a) the co-culture is disposed upon a first surface of the support facing a gaseous phase;
b) a second surface of the support faces and is in contact with the liquid culture medium; and
c) the liquid culture medium is retained in contact with the support and with the culture by virtue of surface tension and/or capillarity.

### Claim 39 (depends on 37)

The co-culture of claim 37, wherein the tumour cells are present in the form of aggregates of at least 5 cells.

### Claim 40

A method of preparing a three-dimensional organotypic cell co-culture comprising the steps of
a) providing a first cell suspension comprising tumour cells and a second cell suspension comprising matrix cells which are distinct from the tumour cells;
b) combining the first and second cell suspensions to provide mixed cells suspended in a liquid suspension medium;
c) concentrating and/or compacting the mixed cells;
d) incubating the mixed cells obtained from step (c) on a first surface of a semi-permeable support facing a gaseous phase, wherein during the incubation a second surface of the support faces and is in contact with a liquid culture medium, and wherein the liquid is retained in contact with the support and the mixed cells by virtue of surface tension and/or capillary action.

### Claim 41 (depends on 40)

The method of claim 40, wherein during the incubation of step (d), the tumour cells grow three-dimensionally disposed within a matrix formed by the matrix cells.

### Claim 42 (depends on 40)

The method of claim 40, wherein providing the first and/or second cell suspensions comprises disassociating cells from a solid tissue source.

### Claim 43 (depends on 40)

The method of claim 40, wherein the mixed cells prior to incubation comprise matrix and tumour cells in a matrix:tumour cell ratio from 100:1 to 10:1.

### Claim 44 (depends on 40)

The method of claim 40, wherein the mixed cells are compacted in step (c) by capillary action, evaporation, centrifugation, gravitation, the application of hydrostatic pressure, pumping, suction, or aspiration.

### Claim 45 (depends on 40)

The method of claim 40, wherein step (c) comprises centrifugation of the mixed cells and removal of supernatant liquid suspension medium.

### Claim 46 (depends on 45)

The method of claim 45, wherein the mixed cells are placed upon the support of step (d) after the centrifugation and removal of supernatant liquid suspension medium of step (c).

### Claim 47 (depends on 45)

The method of claim 45, wherein the mixed cells are placed upon the support of step (d) by the centrifugation.

### Claim 48 (depends on 40)

The method of claim 40, wherein step (d) comprises allowing the cells to further compact upon the support.

### Claim 49 (depends on 48)

The method of claim 48, wherein said further compaction is by evaporation and/or capillary action.

### Claim 50 (depends on 40)

The method of claim 40, wherein the capillary action of step (d) further compacts the mixed cells.

### Claim 51 (depends on 40)

The method of claim 40, wherein the capillary action of step (d) also compacts the mixed cells in step (c).

### Claim 52 (depends on 40)

The method of claim 40, wherein the method further comprises the step of cryopreserving the organotypic co-culture.

### Claim 53 (depends on 38)

The co-culture of claim 38, wherein the second surface of the support is contralateral to the first surface.

### Claim 54 (depends on 37)

The co-culture of claim 37, wherein the matrix cells are non-cancerous cells.

### Claim 55 (depends on 37)

The co-culture of claim 37, wherein the matrix cells comprise more than one cell type.

### Claim 56 (depends on 37)

The co-culture of claim 37, wherein the matrix cells consist of a single cell type.

### Claim 57 (depends on 37)

The co-culture of claim 37, wherein the tumour cells are not genetically engineered.

### Claim 58 (depends on 37)

The co-culture of claim 37, wherein the tumour and/or matrix cells are mammalian.

### Claim 59 (depends on 37)

The co-culture of claim 37, wherein the tumour and/or matrix cells are human.

### Claim 60 (depends on 37)

The co-culture of claim 37, wherein the matrix cells comprise stem cells, primary cells, stromal cells, or cells derived from a source selected from a biopsy, cadavaric tissue, mammalian or human tissue-derived cell lines, the central nervous system, brain, bone marrow, blood, spleen, retina thymus, heart, mammary gland, liver, pancreas, thyroid, skeletal muscle, kidney, lung, intestine, ovary, bladder, testis, uterus or connective tissue.

### Claim 61 (depends on 37)

The co-culture of claim 37, wherein the tumour cells are cancer stem cells

### Claim 62 (depends on 37)

The co-culture of claim 37, wherein the tumour cells are derived from a tumour of stem cells, pancreas, blood, cervix, colon, intestine, kidney, brain, mammary gland, ovary, prostate, skin, liver, lung or spleen.

### Claim 63 (depends on 38)

The co-culture of claim 38, wherein the support comprises hydrophilic PTFE, PET or aluminium oxide.

### Claim 64 (depends on 38)

The co-culture of claim 38, wherein the support is optically transparent.

### Claim 65 (depends on 38)

The co-culture of claim 38, wherein the support is coated with a material that facilitates adhesion of the co-culture to the support.

### Claim 66 (depends on 38)

The co-culture of claim 38, wherein the material coating the support is selected from collagen, laminin or fibronectin.

### Claim 67 (depends on 40)

An in vitro three-dimensional organotypic cell co-culture obtainable by the method of claim 40.

### Claim 68 (depends on 37)

A method for screening for anti-cancer agents, said method comprising contacting the co-culture of claim 37 with a test agent, wherein an inhibitory effect of the test agent upon the growth, proliferation, viability or migration of the tumour cells of the co-culture indicates that the test agent is a candidate anti-cancer agent.

### Claim 69 (depends on 37)

An assay kit comprising one or more co-cultures of claim 37.

### Claim 70 (depends on 37)

A device for screening compounds comprising one or more co-cultures of claim 37.