Upstream bioprocess design for cultured meat production

: (Alternative Format Thesis)

Abstract

Cultured meat as a tissue engineering product, in its simplest form, is skeletal muscle tissue. Cultured meat products have the potential to contribute to global food secu rity by diversifying protein sources in a manner that is healthy for people, humane for livestock animals and sustainable for the environment. Realization of these proposed benefits will depend on the specific production process in question. The aim of this thesis was to design a scalable bioreactor with operating data for myoblast cell expan sion, through the evaluation of different upstream bioprocess design aspects inclusive of suitable scaffolds, medium requirements and bioreactor design and operation. Scaffold materials suitable for the scale-up and subsequent commercialization of cul tured meat need to be cost effective and accessible. Use of decellularized garden-variety grass as an inexpensive and sustainable scaffold was explored, and found to have a stri ated topography that, without the need for functionalization, supports the attachment, proliferation, alignment and differentiation of murine C2C12 myoblasts. Incorporation into bioreactors was investigated and areas for optimization identified. The environmental impact of cultured meat - as modelled by peer-reviewed, anticipa tory life cycle assessments (LCAs) to date - have reported its largest impact is due to the culture medium requirements. Here, experiments using C2C12 myoblasts were conducted in static planar culture (well plates) to determine metabolic consumption and production rates, with higher specific rates found for proliferation than differen tiation. This empirical kinetic data for the metabolism of glucose, lactate and amino acids was used in conjunction with in silico modelling to assess the impact of cell doubling time, doubling viability, specific rates and differentiation mass increase on bioprocess batch times and estimations of the amount of culture medium required to produce 1 kg of wet biomass. This identified the differentiation mass increase as an area of uncertainty with a high potential impact on the culture medium requirements. Experiments to measure the protein increase during differentiation highlighted a linear empirical relationship of 18.23 ± 0.48 % day−1 up to day 9 in differentiation medium. Scale-up remains one of the technical challenges within the field. The use of hollow fiber biroeactors (HFBs), as a potential high cell density bioreactor was investigated. HFBs were characterized for the expansion of myoblasts in terms of operating conditions, cell growth and metabolism kinetics, and found to be comparable to static planar culture. Monitoring of glucose, lactate and dissolved oxygen was used to indirectly track the progress of an HFB run, and this empirical data was used in conjunction with in silico models to inform media feeding strategies. Methods of scaling HFBs by size increase, scale out, and process intensification were demonstrated experimentally, and HFBs were shown to be suitable for the high cell density expansion of C2C12 myoblasts with 1.1 × 108 cells/mlECS volume achieved. Taken together, the findings outlined in this thesis provide a combination of fundamen tal empirical data on myoblast metabolism, evaluation of scaffold suitability, and the characterization of HFBs for expansion, setting the scene for an upstream bioprocess where skeletal muscle cells are the product of interest for cultured meat production.

Date of Award	2 Nov 2022
Original language	English
Awarding Institution	University of Bath
Supervisor	Marianne Ellis (Supervisor) & Paul De Bank (Supervisor)