# US 2018/0023049 A1 · Synthetic peptide hydrogel formulations for use as extracellular matrix

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2018/0023049 A1
- Document ID: US20180023049A1
- Status: Published application
- Kind: A1
- Application number: 15/546,980
- Filed: 2016-02-25
- Published: 2018-01-25
- Earliest claimed: 2015-02-25
- Assignees: 3-D Matrix, Ltd.
- Applicants: 3-D Matrix, Ltd.
- Inventors: Eun Seok Gil; Karl Patrick Gilbert
- Web page: https://patentgrep.com/patent/US20180023049A1

## CPC

- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12N 5/0062: {General methods for three-dimensional culture}
- C12N 2533/50: Proteins
- C12N 2539/00: Supports and/or coatings for cell culture characterised by properties
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Synthetic peptide hydrogel solutions having a pH level of about 3.5 or less and a tonicity within an isotonic osmolality range for use in cell culture experimentation. Related kits and cell culture methods are also disclosed.

## Claims

### Claim 1

A cell culture kit, comprising:
a synthetic peptide hydrogel solution;
a dilution solution; and
instructions to:
adjust a concentration of the synthetic peptide hydrogel solution with the dilution solution while maintaining its tonicity at a plasma osmolality level and its pH at a level of about 3.5 or less, and
conduct a cell culture experiment with the diluted synthetic peptide hydrogel solution.

### Claim 2 (depends on 1)

The kit of claim 1, wherein the synthetic peptide hydrogel comprises:
RADARADARADARADA (RADA16).

### Claim 3 (depends on 1)

The kit of claim 1, wherein the synthetic peptide hydrogel solution is formulated such that a pH level of the synthetic peptide hydrogel solution is about 3.5 or less, and a tonicity of the synthetic peptide hydrogel solution is at a plasma osmolality level prior to dilution.

### Claim 4 (depends on 1)

The kit of claim 1, further comprising a cocktail solution formulated to adjust the pH level of the synthetic peptide hydrogel solution to about 3.5 or less, and the tonicity of the synthetic peptide hydrogel solution to the plasma osmolality level prior to dilution.

### Claim 5 (depends on 4)

The kit of claim 4, wherein at least one of the cocktail solution and the dilution solution comprises one or more isotonic agents to control tonicity.

### Claim 6 (depends on 5)

The kit of claim 5, wherein the one or more isotonic agents include salts, sugars, and mixtures thereof.

### Claim 7 (depends on 3)

The kit of claim 3, wherein at least one of the cocktail solution and the dilution solution comprises one or more alkali salts or acidic salts to control pH.

### Claim 8 (depends on 3)

The kit of claim 3, wherein the cocktail solution is hypertonic and has a pH level of between about 1 and about 14.

### Claim 9 (depends on 1)

The kit of claim 1, further comprising at least one of: a source of cells to be cultured, a cell culture medium, and an isotonic buffer solution.

### Claim 10 (depends on 9)

The kit of claim 9, wherein the isotonic buffer solution comprises PBS, saline, a sugar-based isotonic agent including but not limited to sucrose, or a cell culture medium including but not limited to DMEM.

### Claim 11 (depends on 1)

The kit of claim 1, further comprising a washing solution.

### Claim 12 (depends on 11)

The kit of claim 11, wherein the instructions further provide direction to isolate cultured cells with the washing solution, and to subject the isolated cells to cellular or molecular characterization.

### Claim 13 (depends on 1)

The kit of claim 1, wherein the instructions are directed to a 3-D cell culture protocol in which the plurality of cells is suspended in buffer solution.

### Claim 14 (depends on 1)

The kit of claim 1, wherein the instructions are directed to a 2-D cell culture protocol.

### Claim 15 (depends on 3)

The kit of claim 3, wherein the instructions provide further direction to select the plasma osmolality level based on a target cell type and to use the cocktail solution to achieve the plasma osmolality level in the synthetic peptide hydrogel solution.

### Claim 16

A cell culture method, comprising:
maintaining a pH level of a synthetic peptide hydrogel solution at about 3.5 or less;
maintaining a tonicity of the synthetic peptide hydrogel solution at a plasma osmolality level;
diluting the peptide hydrogel solution to a predetermined concentration;
culturing cells in the peptide hydrogel solution for a predetermined period of time; and
isolating the cultured cells.

### Claim 17 (depends on 16)

The method of claim 16, further comprising subjecting the isolated cells to molecular characterization involving flow cytometry analysis and image observation, cell blotting, or polymerase chain reaction (PCR) testing.

### Claim 18 (depends on 16)

The method of claim 16, wherein the synthetic peptide hydrogel solution comprises:
RADARADARADARADA (RADA16).

### Claim 19 (depends on 16)

The method of claim 16, wherein the culturing step is directed to a 3-D cell culture experiment.

### Claim 20 (depends on 16)

The method of claim 16, wherein the culturing step is directed to a 2-D cell culture experiment.

### Claim 21 (depends on 16)

The method of claim 16, wherein the culturing step is directed to recruiting cells into the hydrogels from surrounding cells in vitro or in vivo.

### Claim 22 (depends on 16)

The method of claim 16, wherein the culturing step is directed to an in vitro cell culture experiment.

### Claim 23 (depends on 16)

The method of claim 16, wherein the culturing step is directed to an ex vivo cell culture experiment.

### Claim 24 (depends on 16)

The method of claim 16, wherein the culturing step is directed to an in vivo animal or human experiment.

### Claim 25 (depends on 23)

The method of claim 23, wherein the culturing step involves combination with drugs, cytokines, growth factors, peptides, and/or proteins to improve cell culture and drive cell growth and differentiation.

### Claim 26 (depends on 24)

The method of claim 24, further comprising a step of selecting the plasma osmolality level based on a target species or a target cell type of cell culture experimentation.

### Claim 27 (depends on 26)

The method of claim 26, wherein the tonicity is in a range of about 260 to about 360 mOsm/L.

### Claim 28

A synthetic peptide hydrogel solution having a pH level of about 1.5 to about 3.5, and a tonicity at plasma osmolality in a range of about 260 to about 360 mOsm/L with respect to a target species and a target cell type.

### Claim 29 (depends on 28)

The solution of claim 28, wherein the target species is selected from the group consisting of but not limited to: mouse, rat, swine, rabbit, bovine, human, insect, bacteria, and plant.

### Claim 30 (depends on 28)

The solution of claim 28, wherein the target cell type is selected from the group consisting of but not limited to: fibroblast, stem cells, epithelial cells, endothelial cells, neural cells, cardiac cells, kidney cells, blood cells, muscle cells, pancreatic cell, immune cells, dendritic cells, epidermal cells, cancer cells, astrocytes, adipocytes, hepatic cells, osteoblasts, and chondrocytes.

### Claim 31 (depends on 28)

The solution of claim 28, wherein the synthetic peptide hydrogel solution comprises:
RADARADARADARADA (RADA16).

### Claim 32 (depends on 28)

The solution of claim 28, wherein the synthetic peptide hydrogel solution is characterized by a storage modulus of at least 5 Pa at 0.5% concentration when measured at 1 rad/sec and 1 Pa of stress.

### Claim 33 (depends on 28)

The solution of claim 28, wherein the synthetic peptide hydrogel solution comprises one or more isotonic agents including salts, sugars, and mixtures thereof to control the tonicity, and one or more alkali salts or acidic salts to control the pH level.