Global food production management has become a challenge with an anticipated population of 10 billion people by 2050 and the ongoing COVID-19 epidemic. Seafood is a vital food source due to its widespread consumption, excellent nutrient profile, and low feed conversion ratio, rendering its sustainable production quintessential. Cellular agriculture or cultured meat can increase seafood production; however, the conventional use of Fetal Bovine Serum (FBS) in culture media restricts its utilization at an industrial level. FBS is effective but has many limitations: unethical animal extraction, high demand and low supply, poorly defined ingredients, variable performance, and high cost that impedes the feasibility and commercial viability of cellular agriculture. Thus, employing serum-free media becomes a quintessential need for cellular agriculture. This project aims to replace or reduce the typical 10% serum usage in Zebrafish embryonic stem cell (ESC) production media with protein hydrolysates derived from low-cost natural sources with high protein content. Enzymatic hydrolysis was performed on nine sources: insects (black army fly and cricket), plants (pea), fungi (mushroom and yeast), algae, and marine invertebrates (oyster, mussel, and lugworm). The resulting hydrolysates were evaluated for serum replacement in zebrafish ESCs. All hydrolysates were used at five different concentrations (10, 1 0.1, 0.01 and 0.001 mg/mL) in serum concentrations of 10%, 5%, and 0% with four biological replicates. The best hydrolysate sources and concentrations were selected for further testing at 2.5% and 1% serum concentrations. All hydrolysates, except for cricket, could restore or significantly increase cell growth with 50% less serum at a concentration of 0.1-0.001mg/mL. Protein hydrolysate concentration of 10 and 1mg/mL was toxic for cells. Additionally, the eight hydrolysates could reduce serum concentrations up to 75–90%. However, no protein hydrolysate could completely replace serum, as cells using only protein hydrolysates exhibited morphological aberrations and decreased growth. Replacing serum with protein hydrolysates lowers cellular agriculture's overall cost, thus enabling the commercialization of cultured meat and the development of a sustainable food system. In the future, blending various protein hydrolysate sources with or without the addition of conventional growth factors could be done to create the ideal serum-free media. / Doctor of Philosophy / With a predicted population of 10 billion by 2050 and the ongoing COVID-19 outbreak, the management of global food production has become a dilemma. However, due to its widespread consumption and good nutrient profile, seafood is an essential food supply, making its sustainable production indispensable. Both capture fisheries and aquaculture are conventional ways to produce seafood. However, they are under tremendous pressure and require alternatives that can alleviate this demand and contribute to the sustainable growth of seafood. In-vitro cultured meat, also known as lab-grown meat, is a novel technique with the potential to supplement the traditional fish sector. It appears a great option, as it completely imitates meat and offers numerous environmental, financial, and health advantages. A culture medium supports the existence, survival, growth, and multiplication of meat-producing cells and tissues in cell-based meat. However, the culture medium uses a Fetal Bovine Serum (FBS) supplement, which dramatically increases the cost and raises many ethical concerns as it is derived from a cow's fetus. In this thesis, we substitute FBS with protein hydrolysates derived from nine distinct sources. Hydrolysing proteins with enzymes produce protein hydrolysates, rich in nutrients and peptides that promote cell development. Enzymes were used to hydrolyse nine unique and protein-rich sources, including insects (black army fly and cricket), plants (pea), fungi (mushroom and yeast), algae, and marine creatures (oyster, mussel, and lugworm). The resultant hydrolysates were investigated for replacement of serum in cell culture. Eight protein hydrolysates successfully replaced 90% of serum without impairing cell growth and structure but could not replace serum entirely. In the future, serum-free media could be created by combining these various protein hydrolysates with or without adding other growth-promoting components.

Replacing meat from animals with cultured meat and edible insects might reduce the impact on climate and the environment. That said, in order to fulfill their function as a sustainable food, insects and cultured meat would need to be consumed, on a wider scale and with a real-time reduction in meat consumption.   The aim of this study was to explore consumers' attitudes toward consuming insects and cultured meat, whilst gathering an understanding of what consumers, themselves, believe to be acceptable to consume in the future. Fifteen individual semi-structured interviews, of both men and women, were conducted outside a supermarket in central Sweden. Data was transcribed and analyzed thematically. The results highlight that for some respondents, insects are perceived as alien and undesirable, while cultured meat was perceived as too unnatural to be eaten; whilst others expressed both reluctance and a curiosity towards cultured meat and edible insects. Most respondents believed they will eat less meat, more vegetables and vegetarian protein in the future, and might try insects and cultured meat in hidden forms such as in sausage. The conclusion was that consumers had a mixed attitude toward eating insects and cultured meat. / Enligt FN och FAO måste matvanorna i västvärlden ändras eftersom de inte är hållbara. Jordbruket orsakar utsläpp av stora mängder växthusgaser, och köttproduktionen står för den allra största delen av dessa. Ätbara insekter och odlat kött kan vara alternativa proteinkällor till exempelvis kött, de är mer miljövänliga med avseende på koldioxidutsläpp, vattenförbrukning, markanvändning och energiåtgång. För att livsmedlen ska fylla sin funktion som hållbara proteinkällor så krävs det att de konsumeras av befolkningen. Syftet med denna studie var att studera svenska konsumenters attityder till att äta insekter och odlat kött, samt att undersöka vad konsumenterna själva tror att de kommer att äta i framtiden.   Studien är kvalitativ med en fenomenologisk ansats. Data insamlades med hjälp av intervjuer. 15 personer i olika åldrar deltog i studien, varav 8 kvinnor och 7 män.   Resultatet visade att det finns en blandad attityd till att äta insekter och odlat kött. Insekter upplevdes som främmande och äckligt, och odlat kött som alltför onaturligt för att ätas. Det fanns både en tveksamhet och en nyfikenhet hos respondenterna till dessa alternativa proteinkällor. Hos de som hade en positiv attityd till att äta livsmedlen uppgavs miljövinsterna som främsta anledning till att konsumera dem. Hos de som hade en negativ attityd angavs känslor av äckel som främsta anledning till att inte konsumera insekter och odlat kött. Om insekterna eller det odlade köttet var dolt såsom i korv kunde vissa respondenter tänka sig att äta. Faktorer som ett fördelaktigt pris, god tillgänglighet och att produkter baserade på odlat kött och insekter är godkända enligt livsmedelslagstiftning, påverkade respondenternas villighet att konsumera dessa. Respondenterna trodde framförallt att de kommer att äta mindre kött, mer grönsaker och mer vegetariskt protein i framtiden.   Slutsatsen är att det finns både positiva och negativa attityder till att äta insekter och odlat kött. Viljan att konsumera produkter baserade på dessa livsmedel verkar öka om livsmedlet är dolt, såsom i korv. Detta gäller både för insekter och odlat kött.

Studien undersöker människors inställning till att äta laboratorieodlat kött samt hur produktionen i laboratorium påverkar klimatet jämfört med konventionell produktion. Vi vill förstå vilka faktorer som påverkar inställningen till att konsumera laboratorieodlat kött och vad som ligger bakom den. Tre fokusgrupper genomfördes med hjälp av en guide som utformades innan genomförandet. De tre grupperna var baserade på deltagarnas kosthållning, då det skulle finnas en blandning av olika kosthållningar representerade i grupperna. Vi valde att låta både köttätare och icke-köttätare delta för att se hur synen på laboratorieodlat kött skiljde dom åt. Fokusgruppsdiskussioner transkriberades och delades upp i teman för att sedan analyseras med hjälp av en modell baserad på miljöetik, theory of planned behaviour, kognitiv dissonans och attityder. Fokusgrupperna visade att deltagarnas inställning till laboratorieodlat kött är att det är främmande, äckligt, obehagligt men spännande. Vi kom fram till att de påverkande faktorerna är generation, kön, ekonomi, kosthållning och hälsa. Det är alla faktorer som i sin tur påverkas av normer, attityder, kunskap och miljöetik. / The aim of this study is to find out people's attitude towards consuming cultured meat and how the production in the laboratory affects the climate compared to conventional production. We want to understand which factors are being behind the attitudes towards cultured meat, and how they affect the attitudes. Three focus groups were held based on a guide designed before the implementation. The three groups consisted of four persons based on diet, creating a mixture of different diets being represented in the groups. We chose to include both meat eaters and non-meat eaters to see how the attitudes towards cultured meat differed. The focus group discussions were transcribed and divided into topics, then analyzed using a model based on environmental ethics, the theory of planned behaviour, cognitive dissonance and attitudes. The focus groups showed that attitudes towards cultured meat is that it is foreign, disgusting, unpleasant but exciting. We stated that the influencing factors are generation, sex, economy, diet and health. These are all factors that are influenced by norms, attitudes, knowledge and environmental ethics.

<p dir="ltr">The goal of this work is to enhance acceptance of cultured meat by replicating traditional meat behavior. This research focuses on the design, manufacture, and mechanics of scaffolds, which provide essential structural support for cell alignment and tissue formation in cultured meat. The integration of biopolymer scaffolds in the final product necessitates a thorough understanding of the thermo-mechanical behavior of scaffolding materials during critical stages like culturing and cooking. Two primary studies were conducted using additive manufacturing processes: (1) vat polymerization and (2) fused filament fabrication. A novel sustainable photocurable soy-based resin was evaluated for high-temperature degradation. The key challenge was ensuring printability and understanding the curing kinetics of the soy resin. For fused filament fabrication, the approach employed an innovative manufacturing-for-design paradigm aimed at mimicking the contraction properties of meat during cooking. The geometry, material properties, and printing process gave rise to a class of novel metamaterials with tunable negative thermal expansions. The key results showed that the thermo-mechanical behavior of soy-based scaffolds could be assessed without the need for costly and time-consuming culturing, as heat did not compromise structural integrity. Furthermore, cooking semi-crystalline biopolymers in an aqueous environment led to material crystallization, which altered the expected deformation mechanisms in the scaffolds. This unexpected behavior was captured in an analytical model accounting for non-linear material properties and print process parameters. By understanding the impact of manufacturing techniques on scaffold behavior, this work established a critical process-property-performance relationship for cultured meat production.</p>

Cultured meat offers a sustainable alternative to traditional meat production, addressing critical environmental and ethical issues. A key aspect of this process is the large-scale proliferation of muscle satellite cells, which can further proliferate and form the primary component of cultured meat. However, ensuring efficient cell proliferation within bioreactors is a significant challenge. Without effective and robust cell proliferation, it would be impossible to meet the production demands of cultured meat. Moreover, 3D cell spheroids tend to form tightly packed structures. As these spheroids grow larger, the limited penetration of oxygen and nutrients can lead to the formation of necrotic cores or cause cells in the central layers to experience cell cycle arrest, resulting in either irreversible senescence or reversible quiescence. This adds complexity to maintaining stable cell growth. To address these challenges, different ECM components, specifically Matrigel and Collagen I, were introduced to alter matrix stiffness and growth factor concentrations. The goal was to address issues of reduced cell proliferation and cell cycle arrest. Results demonstrated that in the absence of ECM, 3D-cultured bovine muscle satellite cells spontaneously formed myospheres but exhibited cell cycle arrest and inhibited proliferation. These issues were reversed with the addition of ECM. Increasing ECM stiffness, particularly through higher concentrations of Matrigel and Collagen I, significantly enhanced cell spreading but had a complex effect on cell proliferation. While Matrigel promoted both cell spreading and proliferation, higher stiffness and growth factor levels were associated with reduced proliferation rates, indicating a trade-off between these processes. Notably, a stiffness of 1.5 Pa with 1.56 mg/ml Matrigel yielded the highest proliferation rate, suggesting this condition might be optimal for use in bioreactor systems. Additionally, increasing matrix stiffness using Collagen I also enhanced cell spreading, indicating that cell spreading is strongly influenced by ECM stiffness. Furthermore, Matrigel reduced the expression of quiescence and senescence markers, helping to maintain cells in a proliferative state. These findings underscore the importance of optimizing ECM properties to balance cell proliferation and structural organization in 3D cultures, providing a foundation for scaling up 3D culture systems in bioreactor settings—a critical step toward large-scale cultured meat production. / Thesis / Master of Applied Science (MASc)

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