Scale-up of an intensified bioprocess for the expansion of bovine adipose-derived stem cells (bASCs) in stirred tank bioreactors.

Cultivated meat is an emerging field, aiming to establish the production of animal tissue for human consumption in an in vitro environment, eliminating the need to raise and slaughter animals for their meat. To realise this, the expansion of primary cells in a bioreactor is needed to achieve the high cell numbers required. The aim of this study was to develop a scalable, microcarrier based, intensified bioprocess for the expansion of bovine adipose-derived stem cells as precursors of fat and muscle tissue. The intensified bioprocess development was carried out initially in spinner flasks of different sizes and then translated to fully controlled litre scale benchtop bioreactors. Bioprocess intensification was achieved by utilising the previously demonstrated bead-to-bead transfer phenomenon and through the combined addition of microcarrier and medium to double the existing surface area and working volume in the bioreactor. Choosing the optimal time point for the additions was critical in enhancing the cell expansion. A significant fold increase of 114.19 ± 1.07 was obtained at the litre scale in the intensified bioprocess compared to the baseline (**p < .005). The quality of the cells was evaluated pre- and post-expansion and the cells were found to maintain their phenotype and differentiation capacity.

Bioprocess development for scalable production of cultivated meat.

Traditional farm-based products based on livestock are one of the main contributors to greenhouse gas emissions. Cultivated meat is an alternative that mimics animal meat, being produced in a bioreactor under controlled conditions rather than through the slaughtering of animals. The first step in the production of cultivated meat is the generation of sufficient reserves of starting cells. In this study, bovine adipose-derived stem cells (bASCs) were used as starting cells due to their ability to differentiate towards both fat and muscle, two cell types found in meat. A bioprocess for the expansion of these cells on microcarriers in spinner flasks was developed. Different cell seeding densities (1,500, 3,000, and 6,000 cells/cm2 ) and feeding strategies (80%, 65%, 50%, and combined 80%/50% medium exchanges) were investigated. Cell characterization was assessed pre- and postbioprocessing to ensure that bioprocessing did not negatively affect bASC quality. The best growth was obtained with the lowest cell seeding density (1,500 cells/cm2 ) with an 80% medium exchange performed (p < .0001) which yielded a 28-fold expansion. The ability to differentiate towards adipogenic, osteogenic, and chondrogenic lineages was retained postbioprocessing and no significant difference (p > .5) was found in clonogenicity pre- or postbioprocessing in any of the feeding regimes tested.

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.

Development of a Chemically Defined Medium for in vitro Expansion of Primary Bovine Satellite Cells.

The use of fetal bovine serum (FBS) in animal cell culture media is widely spread since it provides a broad spectrum of molecules that are known to support cell attachment and growth. However, the harvest and collection procedures of FBS raise ethical concerns and serum is an ill-defined and expensive component. This is especially problematic when it comes to regulatory approval for food applications like cultured meat. The aim of this study is to develop a chemically defined, cost efficient serum-free and animal-free medium that supports the attachment and expansion of bovine myoblasts while maintaining their differentiation capacity. Bovine satellite cells were harvested and isolated from a fresh sample of skeletal muscle tissue and cultured in planar systems. The efficacy of the tested formulations was assessed with metabolic assays and cell counting techniques. Optical microscopy was used to observe cellular morphology and statistical analysis was applied. Based on a comprehensive literature analysis, a defined serum-free medium (SFM) composition was developed consisting of DMEM/F12 as basal medium, supplemented with L-ascorbic acid 2-phosphate, fibronectin, hydrocortisone, GlutaMAX™, albumin, ITS-X, hIL-6, α-linolenic acid, and growth factors such as FGF-2, VEGF, IGF-1, HGF, and PDGF-BB. To our knowledge, this is the first defined serum-free and animal free medium formulation specific for bovine myoblasts to date. We conclude that the SFM formulation supported exponential cell growth up to 97% of the serum-containing golden standard growth medium. All reagents used in this study are chemically defined.

Muscle-derived fibro-adipogenic progenitor cells for production of cultured bovine adipose tissue.

Cultured meat is an emergent technology with the potential for significant environmental and animal welfare benefits. Accurate mimicry of traditional meat requires fat tissue; a key contributor to both the flavour and texture of meat. Here, we show that fibro-adipogenic progenitor cells (FAPs) are present in bovine muscle, and are transcriptionally and immunophenotypically distinct from satellite cells. These two cell types can be purified from a single muscle sample using a simple fluorescence-activated cell sorting (FACS) strategy. FAPs demonstrate high levels of adipogenic potential, as measured by gene expression changes and lipid accumulation, and can be proliferated for a large number of population doublings, demonstrating their suitability for a scalable cultured meat production process. Crucially, FAPs reach a mature level of adipogenic differentiation in three-dimensional, edible hydrogels. The resultant tissue accurately mimics traditional beef fat in terms of lipid profile and taste, and FAPs thus represent a promising candidate cell type for the production of cultured fat.

Needle to needle robot-assisted manufacture of cell therapy products.

Advanced therapeutic medicinal products (ATMPs) have emerged as novel therapies for untreatable diseases, generating the need for large volumes of high-quality, clinically-compliant GMP cells to replace costly, high-risk and limited scale manual expansion processes. We present the design of a fully automated, robot-assisted platform incorporating the use of multiliter stirred tank bioreactors for scalable production of adherent human stem cells. The design addresses a needle-to-needle closed process incorporating automated bone marrow collection, cell isolation, expansion, and collection into cryovials for patient delivery. AUTOSTEM, a modular, adaptable, fully closed system ensures no direct operator interaction with biological material; all commands are performed through a graphic interface. Seeding of source material, process monitoring, feeding, sampling, harvesting and cryopreservation are automated within the closed platform, comprising two clean room levels enabling both open and closed processes. A bioprocess based on human MSCs expanded on microcarriers was used for proof of concept. Utilizing equivalent culture parameters, the AUTOSTEM robot-assisted platform successfully performed cell expansion at the liter scale, generating results comparable to manual production, while maintaining cell quality postprocessing.

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.

Biocompatibility Assessment of Conducting PANI/Chitosan Nanofibers for Wound Healing Applications.

As electroactive polymers have recently presented potential in applications in the tissue engineering and biomedical field, this study is aiming at the fabrication of composite nanofibrous membranes containing conducting polyaniline and at the evaluation of their biocompatibility. For that purpose, conducting polyaniline⁻chitosan (PANI/CS) defect free nanofibres of different ratios (1:3; 3:5 and 1:1) were produced with the electrospinning method. They were characterized as for their morphology, hydrophilicity and electrical conductivity. The membranes were then evaluated for their cellular biocompatibility in terms of cell attachment, morphology and cell proliferation. The effect of the PANI content on the membrane properties is discussed. Increase in PANI content resulted in membranes with higher hydrophobicity and higher electrical conductivity. It was found that none of the membranes showed any toxic effects on osteoblasts and fibroblasts, and that they all supported cell attachment and growth, even to a greater extent than tissue culture plastic. The membrane with the PANI/CS ratio 1:3 supports better cell attachment and proliferation for both cell lines due to a synergistic effect of hydrophilicity retention due to the high chitosan content and the conductivity that PANI introduced to the membrane.