Compositions and methods for increasing the culture density of a cellular biomass within a cultivation infrastructure
Provided herein are methods to increase the culture density and/or thickness of a cellular biomass in a cultivation infrastructure, to improve the culture of cells in the absence of serum in a cultivation infrastructure, and to promote anchorage-independent growth of a cellular biomass in a cultivation infrastructure. The methods comprise inhibiting the HIPPO signaling pathway, for example, by activating YAP1, activating TAZ, and/or inhibiting MOB1, LATS1 kinase, LATS2 kinase, WW45, MST1 kinase, and/or MST2 kinase in the cellular biomass. In some embodiments, the cellular biomass is harvested from the cultivation infrastructure for the formulation of cell-based food products or ingredients, such as animal meat manufactured from cells in an ex vivo process or for therapeutic applications such as organ or tissue transplantation or grafting.
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.