ENGINEERING CELL LINES CAPABLE OF PROLIFERATION IN GROWTH FACTOR FREE MEDIA FORMULATIONS
Provided herein are methods of engineering a cell line for reduced dependence on exogenous growth factor(s). In some embodiments, the method includes introducing into a cell one or more of: a polynucleotide comprising a coding sequence of a growth factor ligand; a polynucleotide comprising a coding sequence of a growth factor receptor; or a polynucleotide comprising a coding sequence of an activated downstream growth factor target, and culturing the cells in a cultivation infrastructure.
1 . A method for culturing a bovine cell line in a cell culture media, said method comprising:
(a) seeding an edible cell culture media with an edible, bovine cell line arising from cells transduced with
a polynucleotide comprising a single coding sequence encoding a single growth factor receptor or growth factor, which is an FGFR, a PDGFR, an activated FGFR, or an activated PDGFR, or a combination of solely a coding sequence encoding an FGFR or an activated FGFR and a coding sequence encoding an IGF;
wherein the polynucleotide is engineered to express the coding sequence or the combination of coding sequences in the cell line; and
(b) culturing the cell line in the cell culture media substantially free of exogenous growth factors,
wherein the cell line proliferates in the absence of exogenous growth factors over 72 hours of culture to a cell density equivalent to a cell density over 72 hours of culture time of a comparable cell line not comprising the polynucleotide and cultured in the presence of the exogenous growth factors.
2 . The method of claim 1 , wherein the cells are transduced with only a polynucleotide encoding a single growth factor or growth factor receptor.
3 . The method of claim 1 , wherein the coding sequence encodes an FGFR or activated FGFR.
4 . The method of claim 1 , wherein the coding sequence encodes a PDGFR or activated PDGFR.
5 . The method of claim 1 , wherein the polynucleotide comprises the combination of a coding sequence encoding an FGFR or an activated FGFR and a coding sequence encoding IGF.
6 . The method of claim 1 , wherein the activated FGFR is FGFR3 having an N540K modification, a K650E modification, a myrist modification, or some combination thereof.
7 . The method of claim 1 , wherein the cell line is cultured in an absence of serum.
8 . The method of claim 1 , wherein the cell line is capable of proliferating to a cell density double the seeded cell density over 72 hours of culture time without exogenous growth factors.
9 . The method of claim 1 , wherein the cell line is cultured to a cell density of 1 million cells per milliliter or more.
10 . The method of claim 1 , wherein the coding sequence for the IGF encodes a fusion protein comprising the IGF and a signal peptide, wherein the signal peptide is IL2 signal peptide.
11 . The method of claim 1 , wherein the coding sequence encodes an FGFR and is one of FGFR1, FGFR2, FGFR3, or FGFR4.
12 . The method of claim 1 , wherein the cells of the cell line are myoblasts, myocytes, satellite cells, side population cells, muscle derived stem cells, mesenchymal stem cells, myogenic pericytes, mesoangioblasts, or fibroblasts.
13 . The method of claim 1 , further comprising the step of forming cell line into a cell-based food product suitable for consumption.
14 . A method for culturing an edible, bovine cell line in a cell culture media, said method comprising:
(a) seeding an edible cell culture media with an edible, bovine cell line arising from cells transduced with
a polynucleotide comprising a combination of solely a coding sequence encoding IGF1 and a coding sequence encoding FGF;
wherein the polynucleotide is engineered to express the coding sequences in the cell line; and
(b) culturing the cell line in the cell culture media substantially free of exogenous growth factors,
wherein the cell line proliferates in the absence of the exogenous growth factors over 72 hours of culture to a cell density equivalent to a cell density over 72 hours of culture time of a comparable cell line not comprising the polynucleotide and cultured in the presence of exogenous IGF1 and FGF.
15 . A method for culturing an edible, bovine cell line in a cell culture media, said method comprising:
(a) seeding an edible cell culture media with an edible, bovine cell line arising from cells transduced with
a polynucleotide comprising a single coding sequence of a single growth factor, which encodes FGF2 or FGF3;
wherein the polynucleotide is engineered to express the coding sequence in the cell line; and
(b) culturing the cell line in the cell culture media substantially free of exogenous growth factors, wherein the cell line is capable of proliferating to a cell density higher than the seeded cell density over 72 hours of culture time of a comparable cell line not comprising the polynucleotide.
16 . The method of claim 14 , wherein the cells of the cell line are myoblasts, myocytes, satellite cells, side population cells, muscle derived stem cells, mesenchymal stem cells, myogenic pericytes, mesoangioblasts, or fibroblasts.
17 . The method of claim 14 , further comprising the step of forming the cell line into a cell-based food product suitable for consumption.
18 . The method of claim 15 , wherein the cells of the cell line are myoblasts, myocytes, satellite cells, side population cells, muscle derived stem cells, mesenchymal stem cells, myogenic pericytes, mesoangioblasts, or fibroblasts.
19 . The method of claim 15 , further comprising the step of forming the cell line into a cell-based food product suitable for consumption.
20 . The method of claim 15 , wherein the cells are transduced with only a single type of a growth factor polynucleotide, wherein the growth factor polynucleotide encodes one or more growth factors.