Traditional meat production has major sustainability and ethical concerns. Cultivated meat helps to address these concerns by reducing the need for mass animal farming. Muscle progenitor cells (MPCs) harvested from skeletal muscle are a promising cell source for cultivated meat. While various protocols have been developed for MPC isolation, which protocol is best suited for the cultivated meat industry requires further investigation. Therefore, the purpose of this thesis was to optimize the MPC isolation technique to produce a pure myogenic cell population and provide the cultivated meat industry with standardized procedures for production. For these proof-of-concept experiments, skeletal muscles harvested from the hindlimb muscles of mice were used. Cells were isolated from the harvested muscle then subjected to one of three protocols for MPC enrichment: pre-plating, ice-cold treatment (ICT), or fluorescence activated cell sorting (FACS). The pre-plating and ICT protocols resulted in impure cell populations with few MPCs after one week in culture. Therefore, FACS using two cell-surface markers, NCAM and CD34, was employed as a more specific method for MPC sorting. CD34+NCAM1- cells grew quickly, however, unwanted cell types remained following FACS. In contrast, CD34+NCAM1+ cells had a consistent small, rounded shape and slow proliferation rate. These cells remained viable in culture for several months and had high Pax7 expression, indicating they were a pure population of myogenic cells. CD34+NCAM1+ cells maintained their capacity to differentiate after culturing for an extended period, demonstrating their potential use for cultivated meat production. The results of this study provide a better understanding of the differences between previously published MPC isolation techniques. Future studies will investigate the potential for CD34+NCAM1+ cells to be grown on a larger scale. These experiments provide insight into MPC populations that may exist in livestock species and will help to streamline the early stages of cultivated meat production. / Thesis / Master of Science (MSc) / Traditional meat production is associated with numerous challenges including animal welfare concerns, human health concerns, and harmful environmental consequences. The global population is predicted to reach 9.7 billion by 2050, emphasizing the importance of alternative food sources to meet the increased food demand. Cultivated meat is a promising new protein source, with the intended purpose of providing a sustainable food source with reduced ethical concerns compared to conventional meat. While there are several challenges to overcome throughout the production process, a major consideration in the early stages of cultivated meat production is cell sourcing. Muscle cells harvested from a tissue biopsy are one proposed starting cell source which has the potential to make up most of the tissue in cultivated meat products. This thesis aimed to improve upon previously published protocols used for muscle cell isolation and provide an optimized cell population for use in cultivated meat production. The cell sorting protocol described in this thesis provides a highly efficient technique for muscle cell purification and long-term growth. The resulting cell population has many characteristics that are pertinent to cultivated meat and may advance the early stages of production.

The global demand for animal proteins is projected to rise by 14% by 2030, amplifying the environmental toll of conventional animal-based protein production. Cultivated meat technology can alleviate the growing demand for protein and address the environmental and ethical concerns associated with conventional livestock farming. However, it faces a critical challenge: the high cost of cell culture media, primarily due to the use of Fetal Bovine Serum (FBS). Substituting serum with protein hydrolysates reduces the production expense of cultivated meat products and promotes establishing a sustainable food system. This study explores black soldier fly larvae (Hermetia illucens) as an emerging ethical and cost-effective alternative protein source to replace serum in media, particularly for cultivated meat production. The development of BSFL protein isolate involved defatting the larva, followed by protein extraction. The protein isolate was then hydrolyzed using an enzyme to produce BSFL hydrolysates. The goal was to supplement the protein isolate and hydrolysates with a serum-free media (B8) and determine their efficacy in replacing the 20% serum requirement for the cell culture of Bovine Satellite Cells. The BSFL protein isolate developed had a crude protein content of 80.42% and an amino acid composition conducive to cell proliferation. Experimental concentrations, ranging from 0.006 mg/ml for hydrolysate to 0.06 mg/ml for protein isolate, exhibited enhanced cell growth. Data from dsDNA quantification revealed no significant difference in growth between cells fed serum-containing growth media (BSC-GM) and BSFL protein hydrolysate (BSFLH_1h) over a short-term study. Results from the multi-passage growth study revealed that BSFLH_1h significantly improved cell growth compared to B8 over 4 passages. However, its doubling time was slower than BSC-GM. Additionally, it was observed that the protein isolate and hydrolysate were cytotoxic at higher concentrations. In the future, identifying and removing the cytotoxic compounds can further optimize the media composition. Immunostaining using Pax7 and DAPI identified supplemented media-maintained satellite cell identity of Bovine satellite cells, offering crucial insights into cellular proliferation. Furthermore, since each cell type requires varying serum and nutrients, testing these isolates and hydrolysates on different cell lines can provide better insight into creating a universal serum-free media. / Master of Science in Life Sciences / The global demand for animal proteins is projected to rise by 14% by 2030, amplifying the environmental toll of conventional animal-based protein production. Meat, dairy, aquaculture, and eggs significantly contribute to food-related emissions and occupy a vast portion of global farmland. Cultivated meat production can alleviate the growing demand for protein and address the environmental and ethical concerns associated with conventional livestock farming. Currently, the production of cultivated meat faces a significant hurdle: the high cost of culture media, primarily attributed to the use of Fetal Bovine Serum (FBS). Substituting serum with protein isolates or hydrolysates reduce the production expense of cultivated meat products and promotes a sustainable food system. Protein isolate and hydrolysates derived from black soldier fly larvae (Hermetia illucens) are rich in protein and essential amino acids and can be used as a cost-effective alternative to serum in cell culture media. The protein isolate and hydrolysates derived from BSFL were tested as supplements to a serum-free media (B8) to evaluate their effectiveness in supporting the growth of Bovine Satellite Cells. The protein hydrolysate demonstrated enhanced cell growth at experimental concentrations. However, it could not completely replace serum requirements without slowing cell growth. Despite challenges such as cytotoxicity at higher concentrations, our study suggests that further refinements and application on various cell types can assist in creating a sustainable and affordable serum-free media for cultivated meat production.

No description available.