EDIBLE AND ANIMAL-PRODUCT-FREE MICROCARRIERS FOR ENGINEERED MEAT

• US 20150079238A1
• Filed: 09/15/2014
• Published: 03/19/2015
• Est. Priority Date: 09/13/2013
• Status: Active Grant

• Current Assignee: Fork & Goode, Inc.
• Original Assignee: Brendan Patrick Purcell, Francoise Suzanne Marga, Gabor Forgacs
• Inventors: MARGA, Francoise Suzanne, FORGACS, Gabor, PURCELL, Brendan Patrick, FORGACS, Andras
• Granted Patent: US 9,752,122 B2
• Time in Patent Office: Days
• US Class Current: 426/92
• CPC Class Codes: A23L 13/00 Meat products; Meat meal; Preparation or treatment thereof
• CPC Class Codes: A23P 10/20 Agglomerating; Granulating; Tabletting
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: A23V 2200/264 All vegan ingredients, i.e. all animal product free
• CPC Class Codes: C12N 2500/92 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 5/0658 Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts

Abstract

Edible microcarriers, including microcarrier beads, microspheres and microsponges, appropriate for use in a bioreactor to culture cells that may be used to form a comestible engineered meat product. For example, the edible microcarriers described herein may include porous microcarriers that may be used to grow cells (e.g., smooth muscle cells) and may be included with the cells in the final engineered meat product, without requiring modification or removal of the cells from the microcarriers. In a particular example, the edible microcarriers may be formed of cross-linked pectin, such as pectin-thiopropionylamide (PTP), and RGD-containing polypeptide, such as thiolated cardosin A. Methods of forming edible microcarriers, methods of using the edible microcarriers to make engineered meat, and engineered meat including the edible microcarriers are also described herein.

Claims

1. A method of forming engineered meat, the method comprising:

culturing a plurality of muscle cells on edible and animal-product-free microcarriers in suspension to form a plurality of cellularized microcarriers, wherein the animal-product-free microcarriers comprise a hydrogel of cross-linked pectin and RGD-containing polypeptides;

forming an aggregate of the cellularized microcarriers; and

at least partially fusing the cellularized microcarriers to form a volume of engineered meat.

2. The method of claim 1, wherein culturing comprises culturing a plurality of muscle cells on edible and animal-product-free microcarriers comprising a hydrogel of thiol-modified pectin (PTP) and cardosin.

3. The method of claim 1, wherein culturing comprises culturing a plurality of muscle cells on edible and animal-product-free microcarriers wherein the animal-product-free microcarriers comprise a flavoring, a flavor enhancer, a colorant, a color enhancer, and a nutritional enhancer.

4. The method of claim 1, wherein forming an aggregate of the cellularized microcarriers comprises placing cellularized microcarriers immediately adjacent each other.

5. The method of claim 1, wherein forming the aggregate comprises placing a plurality of cellularized microcarriers adjacently onto a support substrate.

6. The method of claim 1, wherein forming the aggregate comprises placing a plurality of the cellularized microcarriers adjacently onto a planar support substrate;

fusing said plurality of cellularized microcarriers to form a first planar layer; and

stacking more than 5 additional planar layers onto the first planar layer;

wherein at least partially fusing the cellularized microcarriers to form the volume of engineered meat comprises fusing the layers to form the volume of engineered meat.

7. The method of claim 6, wherein stacking more than 5 additional planar layers comprises stacking more than 10 additional planar layers so that a majority of cells within the volume of engineered meat have died after fusion of the additional planar layers is at least partially complete.

8. The method of claim 1, wherein culturing comprises culturing in a bioreactor.

9. The method of claim 1, wherein culturing the plurality of muscle cells comprises culturing a plurality of non-human myocyte cells.

10. The method of claim 1, further comprising freezing said volume of engineered meat.

11. The method of claim 1, wherein forming the aggregate comprises laying a plurality of cellularized microcarriers having a diameter of between about 0.05 mm and about 3 mm adjacently onto a planar support substrate.

12. The method of claim 1, further comprising sequentially adding additional cellularized microcarriers onto the volume of engineered meat and allowing the additional cellularized microcarriers to at least partially fuse with the volume of engineered meat.

13. The method of claim 1, further comprising sequentially adding additional cellularized microcarriers onto the volume of engineered meat and allowing the additional cellularized microcarriers to at least partially fuse with the volume of engineered meat so that a majority of cells within the volume of engineered meat have died after fusion of the additional cellularized microcarriers is at least partially complete.

14. A method of forming engineered meat, the method comprising:

culturing a plurality of muscle cells on edible and animal-product-free microcarriers in suspension to form a plurality of cellularized microcarriers, wherein the animal-product-free microcarriers comprise a hydrogel of thiol-modified pectin (PTP) and cardosin;

forming an aggregate of the cellularized microcarriers; and

at least partially fusing the cellularized microcarriers to form a volume of engineered meat;

adding additional cellularized microcarriers onto the volume of engineered meat and allowing the additional cellularized microcarriers to at least partially fuse with the volume of engineered meat so that a majority of cells within the volume of engineered meat have died after fusion of the additional cellularized microcarriers is at least partially complete.

15. An engineered meat product, the meat product comprising:

a comestible body having a volume formed of a plurality of cellularized microcarriers, wherein each cellularized microcarrier includes an edible microcarrier comprising a hydrogel of a cross-linked pectin and an RGD-containing polypeptide and a plurality of muscle cells attached to the edible microcarrier, further wherein the cellularized microcarriers are at least partially fused to each other.

16. The engineered meat of claim 15, wherein the edible microcarrier comprises cross-linked thiol-modified pectin and cardosin.

17. The engineered meat of claim 15, wherein the plurality of muscle cells comprise smooth muscle cells.

18. The engineered meat of claim 15, wherein the edible microcarrier comprises a flavoring, a flavor enhancer, a colorant, a color enhancer, and a nutritional enhancer.

19. The engineered meat of claim 15, wherein the cellularized microcarriers have a diameter of between about 0.05 mm and about 3 mm.

20. The engineered meat of claim 15, wherein the edible microcarrier is animal-product-free.

21. A method of forming an edible and animal-product-free microcarrier for use as part of an engineered meat product, the method comprising:

cross-linking a pectin and an RGD-containing polypeptide to form a pectin/RGD-containing polypeptide hydrogel; and

forming microbeads from the pectin/RGD-containing polypeptide hydrogel having a diameter of between about 0.05 mm and about 3 mm.

22. The method of claim 21, further comprising lyophilizing the pectin/RGD-containing polypeptide hydrogel.

23. The method of claim 21, wherein cross-linking a pectin and an RGD-containing polypeptide to form a pectin/RGD-containing polypeptide hydrogel comprises cross-linking pectin-thiopropionylamide (PTP) and cardosin.

24. The method of claim 21, further comprising:

creating pectin-thiopropionylamide (PTP) by derivatization of pectin with cystamine, wherein cross-linking the pectin and the RGD-containing polypeptide comprises cross-linking the PTP with the RGD-containing polypeptide.

25. The method of claim 21, further comprising:

thiolating a cardosin polypeptide, wherein cross-linking the pectin and the RGD-containing polypeptide comprises cross-linking the pectin with the thiolated cardosin polypeptide.

26. The method of claim 21, wherein forming microbeads comprises forming porous microbeads.

27. The method of claim 21, wherein forming microbeads comprises forming microbeads having a diameter of between about 1 mm and about 0.1 mm.

28. A method of forming an edible and animal-product-free microcarrier for use in forming an engineered meat product, the method comprising:

creating pectin-thiopropionylamide (PTP) by derivatization of pectin with cystamine;

thiolating a cardosin polypeptide;

cross-linking the PTP and thiolated cardosin to form a hydrogel;

lyophilizing the hydrogel of cross-linked PTP and cardosin; and

forming microbeads from the cross-linked PTP and cardosin.

29. The method of claim 28, further comprising adding one or more of:

a flavoring, a flavor enhancer, a colorant, a color enhancer, and a nutritional enhancer.

30. An edible microcarrier that is free of animal products and that is configured for culturing muscle cells to form an engineered meat product, the edible microcarrier having a body comprising a hydrogel of cross-linked pectin and an RGD-containing polypeptide, wherein the body is between about 3 mm and about 0.05 mm in diameter.

31. The edible microcarrier of claim 30, wherein the pectin comprises thiol-modified pectin (PTP).

32. The edible microcarrier of claim 30, wherein the RGD-containing polypeptide comprises a cardosin.

33. The edible microcarrier of claim 30, wherein the body has a diameter of between about 2 mm and about 0.05 mm in diameter.

34. The edible microcarrier of claim 30, wherein the body has a diameter of between about 1 mm and about 0.1 mm in diameter.

35. The edible microcarrier of claim 30, wherein the body is porous.

36. The edible microcarrier of claim 30, further comprising an additive consisting of one or more of:

a flavoring, a flavor enhancer, a colorant, a color enhancer, and a nutritional enhancer.

37. An edible microcarrier that is free of animal products and that is configured for culturing muscle cells to form an engineered meat product, the edible microcarrier having a body comprising a hydrogel of thiol-modified pectin and cardosin, wherein the body is between about 3 mm and about 0.05 mm in diameter.