HEAT SHOCK INDUCIBLE CELLS AND THEIR MANUFACTURE AND USE
Provided are methods and systems for producing a cell based product for dietary consumption, methods and systems for selecting cell lines with heat shock inducible genes associated with a fat or muscle phenotype, draw and fill methods and systems, methods and systems for a chemostat with cell retention, chicken or bovine cell lines, and edible bovine or avian cell lines obtained from the methods described herein.
1 . A non-human animal cell cultivation method, comprising:
a. cultivating avian, bovine, or non-human mammalian cells comprising a nucleic acid encoding a factor or biomarker associated with a fat or muscle phenotype operably linked to a heat shock promoter in media in a bioreactor for a first time period at a proliferation temperature;
b. exposing the cells to a heat shock temperature for a second time period, thereby inducing expression of the factor or biomarker;
c. separating at least a portion of the heat shocked cells from the media; and
d. harvesting the separated, heat shocked cells for formulation into a comestible food product.
2 . The method of claim 1 , wherein the cells are cultivated at a proliferation temperature between 35° C. and 40° C. to a target cell density.
3 . The method of claim 2 , the cells are cultivated to a target cell density of about 0.1 mil/mL cells to about 70 mil/mL.
4 . The method of claim 1 , wherein the cells are exposed to a heat shock temperature at or between 37° C. and 43° C. and the second time period is about 3 hours, and wherein the heat shock temperature is at least 3° C. higher than the proliferation temperature.
5 . The method of claim 1 , wherein a substantial portion of the cellular population exhibits expression of the nucleic acid operably linked to the heat shock promoter at 43° C.
6 . The method of claim 1 , wherein less than 10% of the cells express the nucleic acid operably linked to the heat shock promoter at or below 37° C.
7 . The method of claim 1 , wherein the cells have a basal doubling time of 24 hours, and wherein the cells return to the basal doubling time within 6 days of exposure to the heat shock temperature.
8 . The method of claim 1 , wherein the heat shock promoter is a HSP1A1 promoter.
9 . The method of claim 1 , wherein the heat shock promoter is homologous to the species of the cells and comprises one or more portions of the heat shock promoter having heat-inducible promoter activity.
10 . The method of claim 1 , wherein the heat shock promoter is modified to exhibit reduced activation by heavy metals or ultraviolet light as compared to a native heat shock promoter.
11 . The method of claim 1 , wherein the factor or biomarker associated with a fat phenotype is PPARG and/or CEBPA or the factor or biomarker associated with a muscle phenotype is PAX7, MYOD, MYH2 and/or MEF2B (MERFs).
12 . The method of claim 1 , wherein step b is carried out in a differentiation system for the second time period, wherein the differentiation system comprises culturing the cells in a tank or in a pipe through which the cells flow.
13 . The method of claim 1 , wherein after step b, the cells are cultivated for a finishing time period, at a proliferation time between 1 and 14 days.
14 . The method of claim 1 , wherein prior to step d, the cells exhibit a 0.5 fold to 30 fold increase in expression of factors or biomarkers associated with a fat or muscle phenotype compared to non-heat shocked cells.
15 . The method of claim 1 , wherein prior to step d, the cells exhibit phenotypic and genotypic characteristics of adipocytes and/or myotubes.
16 . A chicken or bovine fibroblast cell line, characterized by:
a. suspension adaptation;
b. a population doubling level exceeding 100;
c. a doubling time less than 24 hours;
d. a culture density tolerance exceeding 1×10 7 cells/ml;
e. adaptation to serum free media;
f. edibility; and
g. heat shock inducible gene expression.
17 . The cell line of claim 16 , wherein the cell line returns to a doubling time of less than 24 hours 6 days or less after exposure to a heat shock temperature for a time length between about 1 and 6 hours.
18 . The cell line of claim 16 , wherein heat shock inducible gene expression is induced at a temperature at or between 37° C. and 43° C.
19 . The cell line of claim 16 , wherein the inducible gene expression comprises one or more genes associated with a fat or muscle phenotype operably linked to a heat shock promoter.
20 . The cell line of claim 19 , wherein the factor or biomarker associated with a fat phenotype is PPARG and/or CEBPA or the factor or biomarker associated with a muscle phenotype is PAX7, MYOD, MYH2 and/or MEF2B (MERFs).