# US 2020/0399410 A1 · Tuneable Cell Substrates

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- Number: US 2020/0399410 A1
- Document ID: US20200399410A1
- Status: Published application
- Kind: A1
- Application number: 16/772,584
- Filed: 2018-12-14
- Published: 2020-12-24
- Earliest claimed: 2017-12-15
- Assignees: Cambridge Enterprise Limited
- Applicants: Cambridge Enterprise Limited
- Inventors: Chibeza Chintu Agley; Kevin Chalut; Jose Carlos Rebelo da Silva
- Web page: https://patentgrep.com/patent/US20200399410A1

## CPC

- C08F 220/56: Acrylamide; Methacrylamide
- C08F 222/385: {Monomers containing two or more (meth)acrylamide groups, e.g. N,N'-methylenebisacrylamide}
- C12M 3/02: with means providing suspensions
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12N 5/0607: {Non-embryonic pluripotent stem cells, e.g. MASC (induced pluripotent stem cells C12N5/0696)}
- C12N 5/0654: {Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth}
- B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2533/30: Synthetic polymers
- C08F: MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- 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)
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES

## Abstract

This invention relates to a substrate for mammalian cell culture comprising a co-polymer hydrogel comprising monomeric units of acrylamide, bisacrylamide and a coupling compound, such as 6-acylamidohexanoic acid (6AHA). A cell adhesion molecule, such as a component of the extracellular matrix (ECM), is covalently coupled to the coupling compound monomeric units of the co-polymer. The stiffness and cell adhesion molecule density of the substrate can be independently controlled by altering the concentration of acrylamide and coupling compound in the hydrogel, respectively. Substrates and methods and kits for their production are provided, along with cell culture systems and methods of culturing mammalian cells.

## Claims

### Claim 1

A substrate for mammalian cell culture comprising;
(i) a co-polymer hydrogel comprising monomeric units of acrylamide, bisacrylamide, and coupling compound, and
(ii) a cell adhesion molecule said cell adhesion molecule being covalently coupled to coupling compound monomeric units of the co-polymer.

### Claim 2 (depends on 1)

A substrate according to claim 1 having a stiffness of 0.1 kPa-100 kPa.

### Claim 3 (depends on 1, 2)

A substrate according to claim 1 or claim 2 wherein the co-polymer hydrogel comprises 2-30% monomeric units of acrylamide.

### Claim 4

A substrate according to any one of the preceding claims wherein the co-polymer hydrogel comprises 1 to 100 mM monomeric units of the coupling compound.

### Claim 5 (depends on 4)

A substrate according to claim 4 wherein the co-polymer hydrogel comprises 40-70 mM monomeric units of the coupling compound.

### Claim 6 (depends on 4)

A substrate according to claim 4 wherein the co-polymer hydrogel comprises 70-100 mM monomeric units of the coupling compound

### Claim 7

A substrate according to any one of the preceding claims wherein the substrate has a stiffness of 50 to 100 kPa.

### Claim 8 (depends on 7)

A substrate according to claim 7 wherein the substrate is suitable for culturing osteogenic progenitor cells (OPGs).

### Claim 9 (depends on 1, 2, 3, 4, 5, 6)

A substrate according to any one of claims 1 to 6 wherein the substrate has a stiffness of 5 kPa to 30 kPa.

### Claim 10 (depends on 9)

A substrate according to claim 9 wherein the substrate has a stiffness of 20 kPa to 30 kPa.

### Claim 11 (depends on 9)

A substrate according to claim 9 wherein the substrate has a stiffness of 5 kPa to 15 kPa.

### Claim 12 (depends on 9, 10, 11)

A substrate according to any one of claims 9 to 11 wherein the co-polymer hydrogel comprises 15-20% acrylamide monomeric units

### Claim 13 (depends on 9, 10, 11, 12)

A substrate according to any one of claims 9 to 12 wherein the substrate is suitable for culturing muscle cells.

### Claim 14 (depends on 1, 2, 3, 4, 5, 6)

A substrate according to any one of claims 1 to 6 wherein the substrate has a stiffness of 0.1 kPa to 1.2 kPa.

### Claim 15 (depends on 14)

A substrate according to claim 14 wherein the co-polymer hydrogel comprises 1-10% acrylamide monomeric units.

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

A substrate according to claim 14 or claim 15 wherein the substrate is suitable for culturing stem cells, progenitor cells and brain cells.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

A substrate according to any one of claims 1 to 17 wherein the coupling compound is a compound of formula 1;
wherein
L is selected from optionally substituted C2-10 alkylene, optionally substituted C2-10 alkenylene, —(O—CR2)n— and —(CR2—O)n— wherein n is an integer from 1 to 4;
Q is selected from —C(═O)—, —C(R2)— and —C(R)═C(R)—; and
X is selected from —O—, —S—, —C(R2)—, —C(R)═C(R)—, and —N(R)—.

### Claim 18 (depends on 17)

A substrate according to claim 17 wherein the coupling compound is 6-acylamidohexanoic acid (6AHA).

### Claim 19

A substrate according to any one of the preceding claims wherein the cell adhesion molecule comprises a primary amine group.

### Claim 20

A substrate according to any one of the preceding claims wherein the cell adhesion molecule is an integrin ligand.

### Claim 21

A substrate according to any one of the preceding claims wherein the ECM component is fibronectin.

### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)

A substrate according to any one of claims 1 to 20 wherein the ECM component is laminin.

### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

A mammalian cell culture system comprising;
(a) a substrate according to any one of claims 1 to 22; and
(b) a cell culture medium.

### Claim 24 (depends on 23)

A mammalian cell culture system according to claim 23 wherein the cell culture medium is a defined medium.

### Claim 25 (depends on 23, 24)

A mammalian cell culture system according to claim 23 or claim 24 wherein the cell culture medium is adapted for culture of mammalian stem cells.

### Claim 26 (depends on 23, 24, 25)

A mammalian cell culture system according to any one of claims 23 to 25 further comprising mammalian cells.

### Claim 27 (depends on 26)

A mammalian cell culture system according to claim 26 wherein the mammalian cells are stem or progenitor cells.

### Claim 28 (depends on 27)

A mammalian cell culture system according to claim 27 wherein the stem cells are embryonic stem cells (ESCs), foetal stem cells, adult stem cells, adult progenitor cells, amniotic stem cells, cord stem cells or induced pluripotent stem cells (iPSCs).

### Claim 29 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

A method of culturing mammalian cells comprising;
providing a substrate according to any one of claims 1 to 22;
immersing the substrate in a cell culture medium;
seeding the substrate with mammalian cells; and
culturing the mammalian cells on the substrate.

### Claim 30

A method of producing a cell culture substrate comprising
producing a solution of acrylamide, bisacrylamide and coupling compound monomeric units,
polymerising said monomers to form co-polymer hydrogel comprising acrylamide and coupling compound monomeric units; and
covalently coupling a cell adhesion molecule to the coupling compound monomeric units to produce the cell culture substrate.

### Claim 31 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 30)

A method according to claim 30 wherein the substrate is a substrate according to any one of claims 1 to 22.

### Claim 32 (depends on 30, 31)

A method according to claim 30 or claim 31 wherein the cell adhesion molecule is covalently coupled to the coupling compound monomeric units of the co-polymer hydrogel by a method comprising;
activating the carboxyl groups of the coupling compound monomeric units with one or more activating agents, and
reacting the activated carboxyl groups with a primary amine group of the cell adhesion molecule to form a covalent linkage.

### Claim 33 (depends on 32)

A method according to claim 32 wherein the activating agents are N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC) and N-hydroxysuccinimide (NHS).

### Claim 34 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

A kit comprising a cell culture substrate according to any of claims 1 to 22 or reagents for producing said cell culture substrate.

### Claim 35 (depends on 34)

A kit according to claim 34 wherein the reagents comprise a co-polymer hydrogel comprising monomeric units of acrylamide (A), bisacrylamide and coupling compound, and a cell adhesion molecule.

### Claim 36 (depends on 35)

A kit according to claim 35 wherein the reagents comprise acrylamde, bisacrylamide, coupling compound, one or more polymerisation initiators, and a cell adhesion molecule.

### Claim 37 (depends on 35, 36)

A kit according to claim 35 or claim 36 further comprising one or more activating agents for activating coupling compound monomeric units for coupling to the cell adhesion molecule.

### Claim 38 (depends on 34, 35, 36, 37)

A kit according to any one of claims 34 to 37 further comprising a mammalian cell culture medium.