Granted Patent B2

US 12,709,738 · App. 18/046,083 · Granted Aug 18, 2026

Inventors: Nicholas J. Genovese (Hayward, CA); Meri Teresa Firpo (Oakland, CA); Daphné Dambournet (Berkeley, CA)

Assignee: The Upside Group Inc.

C12N5/0658C07K14/4703C12N5/0656C12N2501/998C12N2511/00

Abstract

Claims (20)

1 . A method for adapting anchorage-dependent cells to suspension culture, comprising:

a) providing anchorage-dependent cells comprising i) a nucleic acid encoding a YAP protein, operably linked to a regulatory sequence that promotes ectopic expression in the anchorage-dependent cells, or ii) a nucleic acid encoding a TAZ protein, operably linked to a regulatory sequence that promotes ectopic expression in the anchorage-dependent cells, wherein the anchorage-dependent cells are poultry or bovine fibroblast cells or poultry or bovine primary muscle-derived cells;

b) ectopically expressing the YAP protein or the TAZ protein in the anchorage-dependent cells in a suspension culture in a cultivation infrastructure which does not comprise a substrate to which cells can adhere, wherein the suspension culture comprises agitation to prevent sedimentation and aggregation, thereby adapting the anchorage-dependent cells to suspension culture to produce suspension-adapted cells which proliferate in the suspension culture;

c) isolating the proliferating suspension-adapted cells from the suspension culture; and

d) culturing the isolated proliferating suspension-adapted cells in a second suspension culture to a density of about 10 5 cells/mL to about 10 10 cells/mL in at least 1000 L.

2 . The method of claim 1 , wherein isolating the proliferating suspension-adapted cells comprises clonal isolation.

3 . The method of claim 1 , wherein the anchorage-dependent cells are myogenic.

4 . The method of claim 1 , further comprising forming the isolated proliferating suspension-adapted cells from step (d) into a comestible food product.

5 . The method of claim 1 , further comprising forming the isolated proliferating suspension-adapted cells in step (d) into a self-adherent aggregate.

6 . The method of claim 5 , wherein the self-adherent aggregate forms from a clonally isolated cell growing in the suspension culture.

7 . The method of claim 1 , further comprising in step (b) inhibiting HIPPO signaling by contacting the anchorage-dependent cells with one or more of lysophosphatidic acid, sphingosine-1-phosphate, and thrombin.

8 . The method of claim 1 , wherein step (b) is in serum free media.

9 . A method for adapting anchorage-dependent cells to suspension culture, comprising:

a) culturing the anchorage-dependent cells in a suspension culture in a cultivation infrastructure which does not comprise a substrate to which the cells can adhere and under conditions that inhibit HIPPO signaling in the anchorage-dependent cells, wherein the suspension culture comprises agitation to prevent sedimentation and aggregation, thereby adapting the anchorage-dependent cells to suspension culture to produce suspension-adapted cells which proliferate in the suspension culture, wherein the anchorage-dependent cells are poultry or bovine fibroblast cells or poultry or bovine primary muscle-derived cells, and wherein inhibiting HIPPO signaling comprises activating YAP and/or TAZ or homologs thereof, or inhibiting MOB1, LATS1 kinase, LATS2 kinase, WW45, MST1 kinase, and/or MST2 kinase or homologs thereof; and

b) passaging the proliferating suspension-adapted cells in the suspension culture to another suspension culture, and growing the cells to a density of about 10 5 cells/mL to about 10 10 cells/mL in at least 1000 L.

10 . The method of claim 9 , further comprising clonally isolating a proliferating suspension-adapted cell from the suspension culture of step (a).

11 . The method of claim 9 , wherein the anchorage-dependent cells have been transfected with a construct comprising a nucleic acid encoding a YAP protein or a construct comprising a nucleic acid encoding a TAZ protein operably linked to a regulatory sequence that promotes ectopic expression in the anchorage-dependent cells, and wherein the YAP protein or the TAZ protein is ectopically expressed in the anchorage-dependent cells in step (a).

12 . The method of claim 11 , wherein the nucleic acid encoding the YAP protein or the nucleic acid encoding the TAZ protein is operably linked to a doxycycline-inducible promoter and the culturing of the anchorage-dependent cells is in the presence of doxycycline.

13 . The method of claim 9 , wherein step (a) is in serum free media.

14 . The method of claim 11 , wherein the regulatory sequence is an inducible promoter.

Assignments (3)

Continuity (3)

References Cited (272)