PhD Thesis Defense
Tuesday, March 5, 2024
Gateway Park, Room GP 1002
1:00-2:00 pm
"Scaling Production of Cultured Meat: Decellularized Plant Suspension Carriers, Satellite Cell Attachment Kinetics Under Shear Stresses, and Algal-Driven Media Recycling"
Richard Thyden '15
Abstract: Current global livestock systems currently 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 yet. 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 which 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 and 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. Next the attachment kinetics of QM7 cells on decellularized spinach was explored. QM7 cells only exhibited greater attachment on 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 in respect to static adhesion time and time under shear stress. Both groups exhibited increased detachment rates given less static adhesion time, or increased time under shear stress. PLL coating only was a significant factor in respect to cell detachment given less than 2 hours of static adhesion time. QM7 cells did not exhibit a reduction in DNA synthesis following exposure to acute shear stress magnitudes of 10 and 60 dyn/cm2. Lastly, we investigated the growth of Chlorella sorokiniana, a thermally resistant microalgal species in spent QM7 cell growth media at 37°C. Algae was grown under variable light intensities with the greatest difference in growth occurring between 13 and 165 µmol/m2/s. Improved growth was observed when C. sorokiniana was grown heterotrophically in the dark, vs. mixotrophically in the light. More rapid algal growth was observed in spent QM7 cell media, when 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 thereby enabling more affordable bioprocesses. Future research into decellularize 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.
Thesis Advisor: Defense Chair:
Dr. Tanja Dominko, DVM, PhD Dr. Catherine Whittington, PhD Professor (Advisor) Assistant Professor (Committee Chair) BME/BBT Department BME Department Worcester Polytechnic Institute Worcester Polytechnic Institute
Defense Committee:
Dr. Pamela Weathers, PhD Dr. Simon Kahan, PhD Professor (Committee Member) President (Committee Member) BBT Department Biocellion Worcester Polytechnic Institute
Dr. Glenn R. Gaudette, PhD Professor (Committee Member) Engineering Department Boston College