# US 2025/0084366 A1 · Supplemented serum-free media including unhydrolyzed plant protein for cultured meat production

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- Number: US 2025/0084366 A1
- Document ID: US20250084366A1
- Status: Published application
- Kind: A1
- Application number: 18/954,053
- Filed: 2024-11-20
- Published: 2025-03-13
- Earliest claimed: 2022-05-20
- Assignees: Trustees of Tufts College
- Applicants: Trustees of Tufts College
- Inventors: David L. Kaplan; Andrew Stout
- Web page: https://patentgrep.com/patent/US20250084366A1

## CPC

- A23L 13/00: Meat products; Meat meal; Preparation or treatment thereof
- C12N 5/0031: {Serum-free culture media}
- A23J 3/14: Vegetable proteins
- C12N 5/0037: {Serum-free medium, which may still contain naturally-sourced components}
- C12N 2500/05: Inorganic components
- C12N 2500/30: Organic components
- C12N 2500/46: Amines, e.g. putrescine
- C12N 2500/76: from plants
- C12N 2501/105: Insulin-like growth factors [IGF]
- C12N 2501/12: Hepatocyte growth factor [HGF]
- C12N 2501/135: Platelet-derived growth factor [PDGF]
- C12N 2501/2306: Interleukin-6 (IL-6)
- C12N 2501/998: Proteins not provided for elsewhere
- C12N 2501/999: Small molecules not provided for elsewhere
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A23J: PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS

## Abstract

A serum-free culture media for expansion of the number of cells and related method of use are disclosed. The disclosed media can be for use in cultured food applications. The disclosed media can include a baseline serum-free and animal component-free culture media and a plant protein composition. The plant protein composition can comprise unhydrolyzed plant proteins. The plant protein composition can comprise plant proteins that are 3 kDa in size or greater. The plant protein composition can be present in the serum-free cell culture media at a final concentration of 0.05 g/L to 1 g/L. The baseline media, in use, provides a baseline growth capability for expansion of the numbers of muscle satellite cells for use in cultured food applications. The disclosed media, in use, provides an improved growth capability for expansion of the number of muscle satellite cells. Methods of making and using the disclosed media are also disclosed.

## Claims

### Claim 1

A serum-free cell culture media for expansion of cells, the media comprising:
a basal culture media; and
a plant protein composition at a final concentration of 0.05 g/L to 50 g/L in the serum-free cell culture media,
wherein the plant protein composition comprises unhydrolyzed plant proteins, and wherein the serum-free cell culture media is free or substantially free of albumin.

### Claim at position 2

2-3. (canceled)

### Claim 4 (depends on 1)

The serum-free cell culture media of claim 1, wherein at least about 70% or 75% of the plant proteins are 10 kDa or greater in size.

### Claim 5 (depends on 1)

The serum-free cell culture media of claim 1, wherein the plant protein composition comprises protein from a plant selected from the group consisting of oilseed crops, non-oilseed oil crops, legumes, pulses, and combinations thereof.

### Claim 6 (depends on 5)

The serum-free cell culture media of claim 5, wherein the plant protein composition comprises protein from an oilseed crop.

### Claim 7 (depends on 1)

The serum-free cell culture media of claim 1, wherein the plant protein composition comprises protein from rapeseed, soybean, sunflower seed, sesame seed, flax seed, camelina, safflower, linseed, grapeseed, pumpkin seed, poppyseed, watermelon seed, and combinations thereof.

### Claim 8 (depends on 1)

The serum-free cell culture media of claim 1, wherein the plant protein composition comprises protein from rapeseed.

### Claim at position 8

9-11. (canceled)

### Claim 12 (depends on 1)

The serum-free cell culture media of claim 1, wherein the basal culture media comprises:
one or more sugars at a concentration of between 0.01 g/L and 10 g/L;
one or more amino acids at a concentration of between 0.001 g/L and 5 g/L;
one or more vitamins at a concentration of between 0.001 g/L and 1 g/L;
one or more trace elements at a concentration of between 0.0001 mg/L and 10 mg/L;
one or more growth-stimulating and/or cell-signaling factors at a concentration of between 0 ng/ml and 100,000 ng/ml; and
one or more transport proteins at a concentration of between 0 g/L and 1 g/L.

### Claim 13 (depends on 1)

The serum-free cell culture media of claim 1, further comprising one or more of the following:
recombinant interleukin 6;
ethanolamine;
curcumin;
oleic acid;
minerals;
hepatocyte growth factor (HGF);
insulin-like growth factor 1 (IGF-1);
platelet-derived growth factor BB (PDGF-BB); and/or
linoleic acid.

### Claim 14

(canceled)

### Claim 15 (depends on 1)

The serum-free cell culture media of claim 1, wherein the plant protein composition is present at a concentration of at least 50 mg/L, at least 100 mg/L, at least 200 mg/L, at least 250 mg/L, at least 500 mg/L, at least 750 mg/L, at least 1 g/L, or at most 50 g/L, at most 25 g/L, at most 15 g/L, at most 10 g/L, at most 5 g/L, at most 2 g/L, at most 1.5 g/L, and at most 1 g/L or any range between these concentrations.

### Claim 16 (depends on 1)

The serum-free cell culture media of claim 1, wherein the plant protein composition is present at a concentration of about 200 mg/L to about 400 mg/L.

### Claim at position 14

17-22. (canceled)

### Claim 23 (depends on 1)

The serum-free cell culture media of claim 1, wherein the basal culture media is selected from the group comprising B8 media, Dulbecco's Modified Eagle Medium (DMEM), Ham's F12 media, Roswell Park Memorial Institute (RPMI) 1640 media, Leibovitz's L15 and a mixture thereof.

### Claim at position 16

24-29. (canceled)

### Claim 30

A method of making a serum-free cell culture media, the method comprising adding a plant protein composition at a final concentration of 0.05 g/L to 50 g/L in the serum-free cell culture media to a basal culture media, thereby producing the serum-free cell culture media.

### Claim at position 18

31-33. (canceled)

### Claim 34

A method of expanding the numbers of or differentiating cells, the method comprising: adding cells to a serum-free cell culture media, incubating the cells in the serum-free cell culture media to allow expansion of the number of or differentiation of cells and production of growth media; and harvesting the cells and/or growth media, wherein the serum-free cell culture media comprises:
a basal culture media; and
a plant protein composition at a final concentration of 0.05 g/L to 50 g/L in the serum-free cell culture media,
wherein the plant protein composition comprises unhydrolyzed plant proteins, and
wherein the serum-free cell culture media is free or substantially free of albumin.

### Claim 35

(canceled)

### Claim 36 (depends on 34)

The method of claim 34, wherein at least about 74% of the plant proteins are 10 kDa or greater in size.

### Claim 37 (depends on 34)

The method of claim 34, wherein the cells are selected from the group consisting of bovine, galline, ovine, porcine, equine, murine, caprine, lapine, and piscine.

### Claim at position 23

38-54. (canceled)

### Claim 55 (depends on 34)

The method of claim 34, wherein the plant protein composition is from a plant selected from the group consisting of oilseed crops, non-oilseed oil crops, legumes, pulses, and combinations thereof.

### Claim 56

(canceled)

### Claim 57 (depends on 34)

The method of claim 34, wherein the plant protein composition is from rapeseed, soybean, sunflower seed, sesame seed, flax seed, camelina, safflower, linseed, grapeseed, pumpkin seed, poppyseed, watermelon seed, and combinations thereof.

### Claim 58 (depends on 34)

The method of claim 34, wherein the plant protein composition is from rapeseed.

### Claim at position 28

59-61. (canceled)

### Claim 62 (depends on 34)

The method of claim 34, wherein the basal culture media comprises:
one or more sugars at a concentration of between 0.01 g/L and 10 g/L;
one or more amino acids at a concentration of between 0.001 g/L and 5 g/L;
one or more vitamins at a concentration of between 0.001 g/L and 1 g/L;
one or more trace elements at a concentration of between 0.0001 mg/L and 10 mg/L;
one or more growth-stimulating and/or cell-signaling factors at a concentration of between 0 ng/ml and 100,000 ng/mL; and
one or more transport proteins at a concentration of between 0 g/L and 1 g/L.

### Claim at position 30

63-65. (canceled)

### Claim 66 (depends on 34)

The method of claim 34, wherein the plant protein composition is present at a concentration of about 200 mg/L to about 400 mg/L.

### Claim at position 32

67-72. (canceled)

### Claim 73 (depends on 34)

The method of claim 34, wherein the basal culture media is selected from the group comprising B8 media, Dulbecco's Modified Eagle Medium (DMEM), F12, and a mixture thereof.

### Claim at position 34

74-81. (canceled)