Development of Biomaterial Scaffolds for Cultured Meat and Engineered Muscle

Student thesis: Doctoral ThesisPhD

Abstract

The sustainability of conventional meat production has long been in question [1]. An alternative may be found in in vitro production of meat from myogenic progenitor cells. A crucial component in this model is a highly renewable, easily processed, edible, and non-animal-derived bioscaffold [2, 3]. This thesis explores the compatibility of a small number of natural biomaterials, mainly plant-based to satisfy the aforementioned criteria, and their development into a bioscaffold on which myogenic cells may be cultured into muscle tissue.
Hydrogel scaffolds were produced from κ-carrageenan by crosslinking aqueous carrageenan solutions using potassium chloride (KCl). The resultant hydrogels were highly susceptible to degradation, and results provide evidence that this is due to displacement of the crosslinking potassium ions by ambient sodium ions. Results showed that the surface coating of these hydrogels imparted stability, particularly when chitosan was used as the coating material. However, coated hydrogels still appeared to lack the ability to support C2C12 cell growth. Another coating material, Bombyx mori silk fibroin (SF), was briefly analysed independently for its biocompatibility with myogenic cells. Films of this silk fibroin were able to support C2C12 proliferation and differentiation. Microfluidic flow focusing was used to produce SF microcarriers. When a mixture of methanol, oleic acid, and span 80 was used as the continuous / outer phase, SF particles with mean diameters as high as 318.87 µm ± 17.89 µm were produced.
Zein was analysed for its ability to support the growth (measured by resazurin metabolic assay), spreading (via image analysis), and differentiation (image analysis after immunostaining for myosin heavy chain) of myogenic cells on zein films alongside tissue culture plastic (TCP) controls. Zein displayed an ability to facilitate these cell functions to a degree comparable to that of TCP Various methods were employed in order to produce scaffolds from zein which would be well-suited for implementation in commonly used bioreactor designs: microfluidic flow focusing and emulsification precipitation for microcarrier scaffolds, as well as wet-spinning into a non-solvent (water) for macrofibre membranes, and electrospinning for nanofibres. The attempts to produce microcarriers and wet-spun fibres were unsuccessful, with the majority of particles exhibiting mean diameters consistently below 50 µm, and fibres failing to precipitate into solid constructs. Electrospun nanofibers were successfully produced, but showed instability in water. Carbodiimide-crosslinking appeared to stabilise the fibres, but reduced their cytocompatibility. Crosslinking via treatment with citric acid also imparted stability, but considerably increased the time taken for production. Allowing the fibres to remain adsorbed to a substrate stabilised the fibres without these drawbacks, and so electrospun zein fibres show great potential as a bioscaffold for cultured meat and wider tissue engineering applications.
Date of Award14 Oct 2020
Original languageEnglish
Awarding Institution
  • University of Bath
SupervisorPaul De Bank (Supervisor) & Marianne Ellis (Supervisor)

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