Kweekvlees uit myoblasten en mesenchymale stromacellen van koeien: mogelijkheden, beperkingen, donor-variatie en het verwijderen van dierlijke componenten uit de kweekmethoden

Animal products and meat in particular are an important part of a versatile human diet from both nutritional and cultural perspectives. However, the production of this important food source is known to have many negative effects on the environment, including high greenhouse gas emissions, deforestation, and water and soil pollution. Therefore, researchers are exploring alternative approaches to manufacture animal products. One of these approaches involves cultivating animal cells outside the animal's body to produce animal proteins and fats. This idea is fundamental for the field called 'cellular agriculture', utilizing cell culture principles previously developed in biomedical sciences. The work presented here explores the potential of two bovine cell types for the production of cultured meat: skeletal muscle-derived myoblasts and adipose tissue-derived mesenchymal stromal cells (AT-MSCs). To be able to meet industrial production goals, the investigated cells should have a high in vitro proliferation (expansion) capacity. Moreover, to replicate the composition and structure of beef, the expanded cells should have high myogenic and adipogenic differentiation capacities. We studied cells from different bovine breeds and ages, tested variations of proliferation and differentiation protocols, including media compositions and substrate coatings. In addition, two animal-free cell culture components were explored in this work: an alternative cell dissociation enzyme and a novel soy-based scaffold. The first chapter focuses on the characterization of primary adult and fetal Belgian Blue myoblasts. We demonstrated that most cell lines from adult animals had a very high proliferation capacity and lacked major chromosomal aberrations after expansion, making them suitable candidates for cultured meat production. Fetal myoblasts had a higher initial myogenic differentiation capacity, but both expanded fetal and adult myoblasts lost their differentiation capacity after expansion. Analysis of transcriptomes and proteomes revealed many downregulated genes and proteins among expanded myoblasts and provided hypotheses to test in future research. The second chapter reports the characterization of fetal, calf, and adult AT-MSCs of two breeds: Belgian Blue and Holstein Friesian. It highlights the importance of selecting the right donor type for a particular application. For cultured meat application, the Holstein Friesian breed stood out because within this breed the cells of calf donors had the highest proliferation capacity and the cells of fetal and adult donors had the highest adipogenic differentiation capacity. The third chapter presents nattokinase as a novel cell dissociation enzyme for the long-term subculture of several cell types: a human embryonic stem cell line, and bovine myoblasts and AT-MSCs. It was tested as a food-grade and animal-free alternative to existing trypsin and TrypLE™ and was found to perform similarly to the existing enzymes, with the advantage of preserving the CD56+ population in cultured myoblasts. The fourth chapter demonstrates the use of a novel scaffold based on soy protein isolate (SPI) for a scalable myoblast culture. The SPI scaffold was able to support myoblast attachment even in the absence of serum. Furthermore, it supported myoblast proliferation and myogenic differentiation on both films and in a scalable microcarrier-based suspension culture. In summary, current doctoral work provides several steps forward in pursuit of scalable and less animal component reliant cultured beef, by informing on selection of primary cell donors, and by offering alternatives to animal-derived cell dissociation enzymes and scaffolds. Additionally, it highlights the limitations of expanded primary myoblasts for differentiation and influence of donor variability on characteristics of myoblasts and AT-MSCs. Future studies need to confirm the results presented in this work with larger sample sizes, control over more donor parameters, and using serum-free medium formulations.