# US 2021/0277360 A1 · Cell linkers for heterotypic cell spheroids

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- Number: US 2021/0277360 A1
- Document ID: US20210277360A1
- Status: Published application
- Kind: A1
- Application number: 17/260,365
- Filed: 2019-07-15
- Published: 2021-09-09
- Earliest claimed: 2018-07-16
- Assignees: Kansas State University Research Foundation; The United States of America, as represented by the Secretary, Department of Health and Human Servic
- Applicants: Kansas State University Research Foundation; The United States of America, as represented by the Secretary, Department of Health and Human Servic
- Inventors: Thu Annelise Nguyen; Ralph E. Parchment; Robert J. Kinders
- Web page: https://patentgrep.com/patent/US20210277360A1

## CPC

- C12N 5/0697: {Artificial constructs associating cells of different lineages, e.g. tissue equivalents (blood vessels C12N5/0691)}
- C07K 5/0817: {the first amino acid being Arg}
- C07K 7/06: having 5 to 11 amino acids
- C12N 5/0062: {General methods for three-dimensional culture}
- C12N 5/0693: {Tumour cells; Cancer cells}
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- G01N 33/5011: {for testing antineoplastic activity}
- G01N 33/5088: {of vertebrates}
- C12N 2502/28: Vascular endothelial cells
- C12N 2502/30: tumour cells
- C12N 2513/00: 3D culture
- C12N 2533/50: Proteins
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- 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)

## Abstract

A three-dimensional heterogenous cell spheroid comprising a plurality of at least two different types of cells and one or more short peptide cell linkers. The peptide cell linkers are linear peptides having respective terminal ends, and comprising an adhesion sequence at or near each end, with a suitable spacer sequence between each adhesion sequence. Methods of making and using such spheroids for in vitro testing are also described.

## Claims

### Claim 1

A three-dimensional heterogenous cell spheroid comprising a plurality of at least two different types of cells and one or more peptide cell linkers.

### Claim 2 (depends on 1)

The spheroid of claim 1, wherein said cells proliferate in said spheroid.

### Claim 3 (depends on 1)

The spheroid of claim 1, wherein each of said peptide cell linkers is a linear peptide having respective terminal ends (an N-terminal end and a C-terminal end), and comprising an adhesion sequence at or near each end.

### Claim 4 (depends on 3)

The spheroid of claim 3, wherein each of said peptide cell linkers further comprises a spacer sequence between each adhesion sequence.

### Claim 5 (depends on 4)

The spheroid of claim 4, wherein each spacer sequence comprises IGPGGDGPGG (SEQ ID NO:2), IGPGPGG (SEQ ID NO:3), or GPGGDGPGG (SEQ ID NO:4).

### Claim 6 (depends on 3)

The spheroid of claim 3, wherein each adhesion sequence comprises YIGSR (SEQ ID NO:1), RGD, KGD, or LDV.

### Claim 7 (depends on 3)

The spheroid of claim 3, wherein the adhesion sequence at one end of said peptide is different from the adhesion sequence at the other end of said peptide.

### Claim 8 (depends on 3)

The spheroid of claim 3, wherein the adhesion sequence at one end of said peptide recognizes a first cell type and the adhesion sequence at the other end of said peptide recognizes a second cell type.

### Claim 9 (depends on 3)

The spheroid of claim 3, wherein said adhesion sequences are each within 5 amino acid resides from their respective terminal end of said peptide.

### Claim 10 (depends on 1)

The spheroid of claim 1, wherein said peptide cell linkers each consist of 25 or less amino acid residues.

### Claim 11 (depends on 1)

The spheroid of claim 1, wherein said different cell types are selected from the group consisting of epithelial cells, stromal cells, endothelial cells, mesenchymal cells, stem cells, tumor cells, fibroblasts, adipocytes, dendritic cells, macrophages, and combinations thereof.

### Claim 12 (depends on 1)

The spheroid of claim 1, said spheroid comprising at least one cancer cell type and at least one non-cancer cell type.

### Claim 13 (depends on 1)

The spheroid of claim 1, wherein said different cell types are uniformly distributed throughout the body of said spheroid.

### Claim 14 (depends on 1)

The spheroid of claim 1, said spheroid having a diameter of from about 50 to about 500 microns.

### Claim 15 (depends on 1)

A composition comprising a hydrogel comprising a plurality of spheroids according to claim 1 distributed therein.

### Claim 16 (depends on 1)

A method of forming a spheroid according to claim 1, said method comprising:
preparing a cell suspension comprising said plurality of at least two different types of cells in a cell culture medium;
mixing said one or more peptide cell linkers with said cell suspension to yield a cell mixture;
adding said cell mixture to a cell culture environment; and
culturing said cell mixture in said cell culture environment under favorable conditions for cell proliferation and spheroid formation in said cell culture environment, wherein each of said spheroids comprises said plurality of at least two different types of cells and one or more peptide cell linkers.

### Claim 17 (depends on 16)

The method of claim 16, wherein said cell culture environment is a hydrogel matrix, said method comprising culturing said cell mixture in said hydrogel matrix under favorable conditions for cell proliferation and spheroid formation in said hydrogel.

### Claim 18 (depends on 17)

The method of claim 17, wherein said adding said cell mixture to a hydrogel matrix comprises:
combining said cell mixture with a peptide suspension comprising a plurality of hydrogel-forming peptides; and
allowing said peptide suspension to form a hydrogel having said cells dispersed therein.

### Claim 19 (depends on 17)

The method of claim 17, wherein the hydrogel-forming peptides are amphiphilic and self-assembling linear peptides.

### Claim 20 (depends on 17)

The method of claim 17, wherein said peptide cell linkers are soluble in said hydrogel matrix.

### Claim 21 (depends on 17)

The method of claim 17, wherein said hydrogel is shear thinning.

### Claim 22 (depends on 17)

The method of claim 17, further comprising collecting said spheroids from said hydrogel matrix.

### Claim 23 (depends on 22)

The method of claim 22, wherein said collecting comprises applying a kinetic force to said hydrogel matrix to reverse said hydrogel and release said spheroids.

### Claim 24 (depends on 1)

An in vitro test method comprising:
providing a spheroid comprising a plurality of at least two different types of cells and one or more peptide cell linkers according to claim 1;
contacting said spheroid with a compound of interest for a period of time; and
detecting the effect of said compound of interest on said spheroid or the cells therein.

### Claim 25 (depends on 24)

The method of claim 24, wherein said spheroid comprises at least one type of cancer cell, wherein said compound of interest is a chemotherapeutic, and wherein at least one effect of said compound of interest on said spheroid is cell death.

### Claim 26 (depends on 25)

The method of claim 25, wherein said cancer cell is collected from a patient and used to form said spheroid prior to said test.

### Claim at position 27

-29. (canceled)

### Claim 30 (depends on 24)

The method of claim 24, said method comprising:
providing a plurality of said spheroids;
contacting said spheroids with a plurality of compounds of interest for a period of time, each of said spheroids being contacted with a respective compound of interest and/or defined combination of compounds of interest from a compound library; and
detecting the effect of said compounds of interest on said spheroids or the cells therein.