| CPC C12N 5/0659 (2013.01) [A23L 13/00 (2016.08); A23L 19/03 (2016.08); A23L 33/105 (2016.08); A23V 2002/00 (2013.01); C12N 2513/00 (2013.01); C12N 2533/90 (2013.01)] | 11 Claims |
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1. A method for producing an in vitro meat, the method comprising:
preparing a non-decellularized plant leaf and incubating the leaf in a cell culture media to obtain a non-decellularized leaf scaffold formed from a spinach leaf;
sterilizing the non-decellularized leaf scaffold with ethanol and a saline rinse;
forming a population of muscle cells by:
extracting a bovine tissue sample;
incubating the sample in a digestion medium to form a tissue pellet;
suspending the pellet in a growth medium to form satellite cells; and
enriching the satellite cells by incubating in a carbon dioxide environment for removing unwanted cells to form a cell suspension including the population of muscle cells;
seeding the non-decellularized leaf scaffold with the population of muscle cells to obtain a leaf scaffold adhered population of muscle cells, seeding further comprising:
coating a cell culture plate with a hydrophobic polymer to obtain a polymer coated plate, the coating formed from polydimethylsiloxane (PDMS);
placing the non-decellularized leaf scaffold on the polymer coated plate;
laying a cloning well on the non-decellularized leaf scaffold;
depositing the population of muscle cells on the non-decellularized leaf scaffold within the cloning well, the hydrophobic polymer and the cloning well maintaining the population of muscle cells in communication with the non-decellularized leaf scaffold;
developing a muscle tissue layer on the leaf scaffold;
assembling a plurality of the muscle tissue layers on the non-decellularized leaf scaffold for exposing the muscle cells to the cell culture media on opposed sides of the muscle tissue layers, further including layering a first leaf scaffold adhered population of muscle cells on a second leaf scaffold for forming an adhered population of muscle cells in a layered three-dimensional support structure of non-decellularized leaf scaffolds;
removing non-adherent muscle cells from the non-decellularized leaf scaffold by washing the leaf scaffold with a buffer to obtain the leaf scaffold adhered population of muscle cells; and
growing the leaf scaffold adhered population of muscle cells in cell culture media for differentiating the leaf scaffold adhered population of muscle cells in a differentiation media, and for achieving an alignment of a kappa value of between 0.17 to 0.45 for myosatellite cells defining the population of muscle cells, thereby obtaining the in vitro meat.
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