Scaling Production of Cultured Meat: Decellularized Plant Suspension Carriers, Satellite Cell Attachment Kinetics Under Shear Stresses, and Algal-Driven Media Recycling

Thyden, Richard

Etd

Scaling Production of Cultured Meat: Decellularized Plant Suspension Carriers, Satellite Cell Attachment Kinetics Under Shear Stresses, and Algal-Driven Media Recycling

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Current global livestock systems contribute a significant portion of protein and calories to the global population; however, they are resource intensive, environmentally taxing, and subject to risk associated with the dynamics of global economics and climate change. Researchers have produced small quantities of skeletal muscle, the primary component of meat, using tissue engineering principles and cells isolated from livestock. This strategy, referred to as “Cellular Agriculture” may help decrease traditional livestock systems. The success of this approach will depend on novel bioprocesses that are capable of sustainably supporting cell populations of unprecedented size. Satellite cells are anchorage dependent skeletal muscle progenitors that are responsible for post-natal muscular growth, maintenance, and recovery. Within non-planar bioreactors, anchorage dependent cells, such as satellite cells, attach to stiff microcarriers and are suspended in culture media. Flow introduces shear stress on the microcarrier surfaces and shear forces have been associated with cell detachment that may be detrimental to proliferation. Additionally, cell culture media is a significant contributor to the high cost of bioprocesses for cellular agriculture. This dissertation sought to develop novel carriers for cultured meat using decellularized plant materials, explore the attachment and adhesion kinetics of satellite cells on different substrates when subject to shear stress, and develop a novel approach to cell culture media recycling using a thermally resistant species of microalgae. First, a rapid, food safe, decellularization procedure was established to yield matrix scaffolds derived from plant tissues and evaluated as cell carriers for lab grown meat. A rapid decellularization protocol (<48hrs) of broccoli florets, corn husks, and jackfruit waste fibers using sodium-dodecyl-sulfate, polysorbate-20, and bleach was established and validated via histology and DNA quantification. Decellularized broccoli carriers were characterized by size and shape. Density measurements were comparable to traditional microcarriers. Satellite cells were inoculated into and cultured within a reactor containing decellularized carriers. Cell adhesion was observed, and cell death was limited. Decellularization decreased the stiffness of all scaffolds. Additionally, cell transfer from scaffold to scaffold (bead-to-bead transfer) was observed on corn husk scaffolds in a dynamic environment. Attachment kinetics of QM7 cells on decellularized spinach was also explored. QM7 cells exhibited enhanced attachment to poly-l-lysine (PLL) coated decellularized plants after two hours. The detachment rates of cells on PLL coated polystyrene and tissue culture treated polystyrene when subject to shear stress were compared to static adhesion time and time under shear stress. Detachment rates decreased based on more static adhesion time, or increased time under shear stress. DNA synthesis did not decrease in QM7 cells after exposure to acute shear stress magnitudes of 10 and 60 dyn/cm2. When Chlorella sorokiniana, a thermally resistant algal species was grown in spent QM7 cell growth media at 37°C, at variable light intensities, best growth occured at 13 µmol/m2/s. Algae grew best heterotrophically in the dark, vs. mixotrophically in the light and growth was faster in spent QM7 cell media, compared to fresh media. Algae removed nearly all glucose and ammonia from spent media within 72hours. No cytotoxic effects were observed on QM7 cells grown in algal-treated growth media. QM7 cells exhibited better metabolic activity in algal-treated spent medium than in untreated spent medium. These results suggest that C. sorokiniana can be grown in spent cell culture media, at 37°C, and potentially extend the lifespan of media, enabling more affordable bioprocesses. Future research into decellularized plant cell carriers, shear stress and cell interactions, and algal-driven culture media recycling systems may aid the realization of cultured meat and ultimately develop a more robust global food economy.

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