Product, system and method of cell cultivation
The present invention provides products, systems, and methods of cultivation of non-human metazoan cells.
1 . A genetically modified non-human metazoan cell line comprising:
a. a gene encoding modified telomerase reverse transcriptase (TERT) or an allelic variant thereof resulting in cell immortalization, wherein the TERT gene is located at a safe harbor site within the genome of the cell; and
b. a genetic modification that alters the level of expression of at least one gene selected from genes encoding fibroblast growth factor (FGF) molecules, resulting in reduced growth factor requirements for the cultivation of non-human metazoan cells compared to wild-type cell lines, wherein the FGF molecule is regulated by a promoter; and
c. adaptation of the cell line to grow in a culture medium comprising a protein hydrolysate at a concentration, expressed as dry protein weight, in a range from 8 g/L to 50 g/L.
2 . The genetically modified non-human metazoan cell line according to claim 1 , wherein the genetically modified non-human metazoan cell line further comprises a transferrin receptor 1 (TFRC) gene encoding a protein having a sequence with at least 80% sequence identity to SEQ ID NO: 12.
3 . The genetically modified non-human metazoan cell line according to claim 1 , wherein the genetically modified non-human metazoan cell line further comprises a transforming growth factor beta receptor 1 (TGFBR1) gene encoding a protein having a sequence with at least 80% sequence identity to SEQ ID NO: 14.
4 . The genetically modified non-human metazoan cell line according to claim 1 , wherein the genetically modified non-human metazoan cell line further comprises a gene encoding an insulin protein having a sequence with at least 80% sequence identity to SEQ ID NO: 9.
5 . The genetically modified non-human metazoan cell line according to claim 1 , wherein the genetically modified non-human metazoan cell line comprises a FGF2 gene encoding a protein having a sequence with at least 80% sequence identity to SEQ ID NO: 11.
6 . The genetically modified non-human metazoan cell line according to claim 1 , wherein the FGF molecule is selected from FGF-2, FGF-5, FGF-1, and FGF-8, and wherein the FGF molecule is regulated by single-stranded DNA or RNA oligonucleotide sequences complementary to a target sequence of mRNA.
7 . The genetically modified non-human metazoan cell line according to claim 1 , wherein the gene encoding the FGF molecule is selected from FGF-2,-FGF-5, FGF-1, and FGF-8, and is regulated by the promoter.
8 . The genetically modified non-human metazoan cell line according to claim 7 , wherein the promoter is at least one selected from glyceraldehyde-3-phosphate dehydrogenase, eukaryotic translation elongation factor 1α, and phosphoglycerate kinase 1.
9 . The genetically modified non-human metazoan cell line according to claim 8 , wherein the promoter of eukaryotic translation elongation factor 1α comprises a sequence of SEQ ID NO: 6.
10 . The genetically modified non-human metazoan cell line according to claim 1 , wherein the genetically modified non-human metazoan cell line has reduced growth factor requirements compared to the wild-type cell line and is adapted for cultivation in a protein-free culture medium.
11 . The genetically modified non-human metazoan cell line according to claim 1 , wherein the genetically modified non-human metazoan cell line further comprises a fusion protein of myr-Akt to reduce growth factor requirements in the culture medium.
12 . The genetically modified non-human metazoan cell line according to claim 1 , wherein the non-human metazoan cell line comprises bovine, avian, porcine, equine, piscine, cervine , or cricetine cell lines.
13 . The genetically modified non-human metazoan cell line according to claim 1 , wherein the protein hydrolysate is prepared from soy, pea, rice, wheat, wheat gluten, corn, faba beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, Spirulina, Chlorella , sunflower, water lentil, mung beans, flax, brewer spent grain, distillers spent grain (DDGS), or tomato pomace.
14 . The genetically modified non-human metazoan cell line according to claim 1 , wherein a total input of amino acids from the protein hydrolysate is at least 75% by weight of a total input of all amino acids in the culture medium.
15 . The genetically modified non-human metazoan cell line according to claim 14 , wherein the culture medium further comprises one or more amino acids added separately, wherein a total input of said separately added amino acids is in a range of 0.02 g/l to 30 g/l.
16 . A genetically modified non-human metazoan cell line comprising:
a. a gene encoding modified telomerase reverse transcriptase (TERT) or an allelic variant thereof, wherein the TERT gene is located at a safe harbor site within the genome of the cell; and
b. adaptation of the cell line to grow in a culture medium comprising protein hydrolysate as a main source of amino acids, wherein the total input of supplemental amino acids added separately to the culture medium comprising the protein hydrolysate is in the range of 0.2% to 25%.
17 . The genetically modified non-human metazoan cell line according to claim 16 , wherein the genetically modified non-human metazoan cell line has a TERT gene located in a safe harbor locus within the bovine phosphodiesterase 4D (bPDE4D) gene on chromosome 20 of the non-human metazoan cell line.
18 . The genetically modified non-human metazoan cell line according to claim 17 , wherein the genetically modified non-human metazoan cell line has a TERT gene located in a safe harbor locus within the bovine phosphodiesterase 4D (bPDE4D) gene on chromosome 20 of the non-human metazoan cell line at position 19533000.
19 . The genetically modified non-human metazoan cell line according to claim 18 , wherein the safe harbor locus within the bovine phosphodiesterase 4D (bPDE4D) gene on chromosome 20 of the non-human metazoan cell line at position 19533000 is in the range of 100,000 bp in both directions from position 19533000 of chromosome 20.
20 . The genetically modified non-human metazoan cell line according to claim 16 , wherein the safe harbor locus is PGRandom site.
21 . The genetically modified non-human metazoan cell line according to claim 16 , wherein the genetically modified non-human metazoan cell line comprises a bovine truncated telomerase reverse transcriptase (rbTERT) gene having a sequence with at least 80% sequence identity to SEQ ID NO: 4.
22 . The genetically modified non-human metazoan cell line according to claim 16 , wherein the genetically modified non-human metazoan cell line comprises a bovine telomerase reverse transcriptase (bTERT) gene encoding a protein having a sequence with at least 80% sequence identity to SEQ ID NO: 1.
23 . The genetically modified non-human metazoan cell line according to claim 16 , wherein the genetically modified non-human metazoan cell line comprises a bovine telomerase reverse transcriptase (bTERT) gene having a sequence with at least 80% sequence identity to SEQ ID NO: 2.
24 . The genetically modified non-human metazoan cell line according to claim 16 , wherein the genetically modified non-human metazoan cell line comprises a bovine truncated telomerase reverse transcriptase (rbTERT) gene encoding a protein having a sequence with at least 80% sequence identity to SEQ ID NO: 3.
25 . The genetically modified non-human metazoan cell line according to claim 16 , wherein an immortalization cassette comprises the gene encoding TERT, which is removed from the cell at the time of inoculation into a cultivation device.
26 . A food product composition comprising genetically modified non-human metazoan cells, wherein the genetically modified non-human metazoan cells comprise:
a. a gene encoding modified telomerase reverse transcriptase (TERT) or an allelic variant thereof resulting in cell immortalization wherein the TERT gene is located at a safe harbor site within the genome of the cell; and
b. adaptation of the cell line to grow in a culture medium comprising protein hydrolysate as a main source of amino acids, wherein at least 75% by weight of the total amino acid input in the culture medium is derived from the protein hydrolysate.
27 . The food product composition according to claim 26 , wherein the genetically modified non-human metazoan cell line comprises a modified Harvey rat sarcoma viral oncogene homolog (HRas) gene comprising SEQ ID NO: 18, wherein the modified HRas gene encodes a modified HRas protein with a Q61L substitution comprising SEQ ID NO: 16.
28 . The food product composition according to claim 26 , wherein the food product is intended for human consumption or as a pet food.
29 . The food product composition according to claim 26 , further comprising at least one additional component selected from saccharides, fats, vitamins, minerals, binders, palatants, antioxidants, colorants and preservatives.
30 . The food product composition according to claim 26 , wherein the composition comprises a cell biomass, wherein the cell biomass has a mass density in a range of 900 kg·m −3 to 1200 kg·m −3 .