Expansion of bovine skeletal muscle stem cells from spinner flasks to benchtop stirred-tank bioreactors for up to 38 days.

Introduction: Successful long-term expansion of skeletal muscle satellite cells (MuSCs) on a large scale is fundamental for cultivating animal cells for protein production. Prerequisites for efficient cell expansion include maintaining essential native cell activities such as cell adhesion, migration, proliferation, and differentiation while ensuring consistent reproducibility. Method: This study investigated the growth of bovine MuSC culture using low-volume spinner flasks and a benchtop stirred-tank bioreactor (STR). Results and discussion: Our results showed for the first time the expansion of primary MuSCs for 38 days in a bench-top STR run with low initial seeding density and FBS reduction, supported by increased expression of the satellite cell marker PAX7 and reduced expression of differentiation-inducing genes like MYOG, even without adding p38-MAPK inhibitors. Moreover, the cells retained their ability to proliferate, migrate, and differentiate after enzymatic dissociation from the microcarriers. We also showed reproducible results in a separate biological benchtop STR run.

Microcarriers for Upscaling Cultured Meat Production.

Due to the considerable environmental impact and the controversial animal welfare associated with industrial meat production, combined with the ever-increasing global population and demand for meat products, sustainable production alternatives are indispensable. In 2013, the world's first laboratory grown hamburger made from cultured muscle cells was developed. However, coming at a price of $300.000, and being produced manually, substantial effort is still required to reach sustainable large-scale production. One of the main challenges is scalability. Microcarriers (MCs), offering a large surface/volume ratio, are the most promising candidates for upscaling muscle cell culture. However, although many MCs have been developed for cell lines and stem cells typically used in the medical field, none have been specifically developed for muscle stem cells and meat production. This paper aims to discuss the MCs' design criteria for skeletal muscle cell proliferation and subsequently for meat production based on three scenarios: (1) MCs are serving only as a temporary substrate for cell attachment and proliferation and therefore they need to be separated from the cells at some stage of the bioprocess, (2) MCs serve as a temporary substrate for cell proliferation but are degraded or dissolved during the bioprocess, and (3) MCs are embedded in the final product and therefore need to be edible. The particularities of each of these three bioprocesses will be discussed from the perspective of MCs as well as the feasibility of a one-step bioprocess. Each scenario presents advantages and drawbacks, which are discussed in detail, nevertheless the third scenario appears to be the most promising one for a production process. Indeed, using an edible material can limit or completely eliminate dissociation/degradation/separation steps and even promote organoleptic qualities when embedded in the final product. Edible microcarriers could also be used as a temporary substrate similarly to scenarios 1 and 2, which would limit the risk of non-edible residues.