Prospective life cycle assessment of a bioprocess design for cultured meat production in hollow fiber bioreactors

The aim of cellular agriculture is to use cell-culturing technologies to produce alternatives to agricultural products. Cul-tured meat is an example of a cellular agriculture product, made by using tissue engineering methods. This study aims to improve the understanding of the potential environmental impacts of cultured meat production by comparing be-tween different bioprocess design scenarios. This was done by carrying out a life cycle assessment (LCA) for a bioprocess system using hollow fiber bioreactors, and utilizing bench-scale experimental data for C2C12 cell prolifer-ation, differentiation and media metabolism. Scenario and sensitivity analyses were used to test the impact of changes in the system design, data sources, and LCA methods on the results to support process design decision making. We com-pared alternative scenarios to a baseline of C2C12 cells cultured in hollow fiber bioreactors using media consisting of DMEM with serum, for a 16-day proliferation stage and 7-day differentiation stage. The baseline LCA used the average UK electricity mix as the energy source, and heat treatment for wastewater sterilization. The greatest reduction in en-vironmental impacts were achieved with the scenarios using CHO cell metabolism instead of C2C12 cell metabolisim (64-67 % reduction); achieving 128 % cell biomass increase during differentiation instead of no increase (42-56 % reduction); using wind electricity instead of average UK electricity (6-39 % reduction); and adjusting the amino acid use based on experimental data (16-27 % reduction). The use of chemical wastewater treatment instead of heat treatment increased all environmental impacts, except energy demand, by 1-16 %. This study provides valuable insights for the cultured meat field to understand the effects of different process design scenarios on environmental impacts, and therefore provides a framework for deciding where to focus development efforts for improving the envi-ronmental performance of the production system.Peer reviewe

Environmental impacts of cultured meat: alternative production scenarios

Cultured meat is produced by culturing animal muscle tissue in a laboratory without growing the whole animals. Its development is currently in a research stage. An earlier study showed that cultured meat production could potentially have substantially lower greenhouse gas emissions, land use and water use compared to conventionally produced meat. The aim of this paper is to amend the previous study by considering alternative production scenarios. The impacts of replacing cyanobacteria based nutrient media with plant based media are assessed. This paper includes more specific modelling of a bioreactor suitable for cultured meat production. Further, this study estimates the water footprint of cultured meat based on a method that is compliant with life cycle assessment. The environmental impacts of cultured meat are compared with conventionally produced meat and with plant based protein sources. It is concluded that regardless of the high uncertainty ranges cultured meat has potential to substantially reduce greenhouse gas emissions and land use when compared to conventionally produced meat.JRC.H.4-Monitoring Agricultural Resource

A Systematically Reduced Mathematical Model for Organoid Expansion

Organoids are three-dimensional multicellular tissue constructs. When cultured in vitro, they recapitulate the structure, heterogeneity, and function of their in vivo counterparts. As awareness of the multiple uses of organoids has grown, e.g. in drug discovery and personalised medicine, demand has increased for low-cost and efficient methods of producing them in a reproducible manner and at scale. Here we focus on a bioreactor technology for organoid production, which exploits fluid flow to enhance mass transport to and from the organoids. To ensure large numbers of organoids can be grown within the bioreactor in a reproducible manner, nutrient delivery to, and waste product removal from, the organoids must be carefully controlled. We develop a continuum mathematical model to investigate how mass transport within the bioreactor depends on the inlet flow rate and cell seeding density, focusing on the transport of two key metabolites: glucose and lactate. We exploit the thin geometry of the bioreactor to systematically simplify our model. This significantly reduces the computational cost of generating model solutions, and provides insight into the dominant mass transport mechanisms. We test the validity of the reduced models by comparison with simulations of the full model. We then exploit our reduced mathematical model to determine, for a given inlet flow rate and cell seeding density, the evolution of the spatial metabolite distributions throughout the bioreactor. To assess the bioreactor transport characteristics, we introduce metrics quantifying glucose conversion (the ratio between the total amounts of consumed and supplied glucose), the maximum lactate concentration, the proportion of the bioreactor with intolerable lactate concentrations, and the time when intolerable lactate concentrations are first experienced within the bioreactor. We determine the dependence of these metrics on organoid-line characteristics such as proliferation rate and rate of glucose consumption per cell. Finally, for a given organoid line, we determine how the distribution of metabolites and the associated metrics depend on the inlet flow rate. Insights from this study can be used to inform bioreactor operating conditions, ultimately improving the quality and number of bioreactor-expanded organoids

Hollow-fiber membrane technology: Characterization and proposed use as a potential mimic of skin vascularization towards the development of a novel skin absorption in vitro model

Dermal bioavailability is currently estimated through skin penetration studies using ex vivo models, which lack any measure of capillary bed function, and thus do not fully reproduce physiological conditions. We propose a novel strategy to mimic skin vascularization using newly fabricated hollow fibers made from a biocompatible membrane material, polystyrene, which is hydrophobic if left untreated, or hydrophilic when its surface polarity is modified through plasma-treatment. Caffeine has been well studied in skin penetration assays and was used here to determine the permeation properties of the hollow fibers in a novel jacketed glass bioreactor. For hydrophobic fibers, approximately 87.2 % of the caffeine dose did not penetrate the porous surface; 0.2 % of the dose was collected after 24 h (permeated through the pores), and therefore 12.6 % of the initial dose was suspected to block the membrane. For hydrophilic fibers, both the percentage of the initial dose that permeated and that of blocking caffeine increased to 1.2 % and 35.2 % respectively. It was concluded that caffeine permeated the hollow fibers at similar times of clearance to those observed in vivo, and therefore shows that this new model could provide a surrogate for capillary-based clearance in in vitro skin absorption studies

Surfactant-free poly(lactide-co-glycolide) honeycomb films for tissue engineering: relating solvent, monomer ratio and humidity to scaffold structure

International audienceOne-step surfactant-free, water-droplet templating has been developed as a fabrication method for a poly(lactide-co-glycolide) (PLGA) film that can be used as a model to investigate the relationship between solvent, monomer ratio, polymer concentration and humidity on its structure. The resulting material is a honeycomb-structured film. Formation of this structure was highly sensitive to solvent, monomer ratio, polymer concentration and humidity. Surfactant-free, water-droplet templating thus allows investigation of fabrication parameters and that PLGA monomer ratio selection is important for scaffold structure but not for MG63 cell attachment and proliferation

Simvastatin release from poly(lactide-co-glycolide) membrane scaffolds

Statins, a group of potent inhibitors of 3-hydroxy-3-methylglutaryl Coenzyme A reductase in cholesterol biosynthesis pathway, have been widely used as a cholesterol lowering drug. The plieotrophic effect of statins on bone metabolism in long-term usage has been begun to be studied during recent years and several in vitro and in vivo studies have demonstrated the ability of statins to promote expression of bone morphogenetic protein-2 (BMP-2), inhibition of osteoclast differentiation and reduction of osteoporotic fractures risk. The high liver specificity and low oral bioavailability of statins, leading to poor peripheral distribution, are the main obstacles to benefit anabolic effects of hydrophobic statins on bone formation. Therefore, developing new administration roots for direct delivery to achieve optimum concentration in the bone microenvironment is of interest. Here we present and compare two approaches of combining statins with bone tissue engineering scaffolds. Simvastatin was combined with a poly(lactide-co-glycolide) (PLGA) membrane scaffold for diffusion-controlled release by dissolving simvastatin (dis-sim) in the membrane casting dope, and for degradation-controlled release by covalently bonding saponifiedsimvastatin (sap-sim) to the PLGA in the spinning dope. Rheological and concentration-dependent membrane morphology changes were observed with saponifiedsimvastatin, suggesting ester bond cleavage and covalent bonding of the statin to the PLGA, but not with dissolved simvastatin. Dissolved simvastatin membranes showed a logarithmic decay release profile while the saponifiedsimvastatin membranes showed constant release. It can be concluded that the covalent bonding of simvastatinto PLGA scaffolds is showing potential for use as a controlled releasescaffold for bone tissue engineering

Novel in vitro and mathematical models for the prediction of chemical toxicity

The focus of much scientific and medical research is directed towards understanding the disease process and defining therapeutic intervention strategies. Whilst the scientific basis of drug safety has received relatively little attention, despite the fact that adverse drug reactions (ADRs) are a major health concern and a serious impediment to development of new medicines. Toxicity issues account for ~21% drug attrition during drug development and safety testing strategies require considerable animal use. Mechanistic relationships between drug plasma levels and molecular/cellular events that culminate in whole organ toxicity underpins development of novel safety assessment strategies. Current in vitro test systems are poorly predictive of toxicity of chemicals entering the systemic circulation, particularly to the liver. Such systems fall short because of 1) the physiological gap between cells currently used & human hepatocytes existing in their native state, 2) the lack of physiological integration with other cells/systems within organs, required to amplify the initial toxicological lesion into overt toxicity, 3) the inability to assess how low level cell damage induced by chemicals may develop into overt organ toxicity in a minority of patients, 4) lack of consideration of systemic effects. Reproduction of centrilobular & periportal hepatocyte phenotypes in in vitro culture is crucial for sensitive detection of cellular stress. Hepatocyte metabolism/phenotype is dependent on cell position along the liver lobule, with corresponding differences in exposure to substrate, oxygen & hormone gradients. Application of bioartificial liver (BAL) technology can encompass in vitro predictive toxicity testing with enhanced sensitivity and improved mechanistic understanding. Combining this technology with mechanistic mathematical models describing intracellular metabolism, fluid-­‐flow, substrate, hormone and nutrient distribution provides the opportunity to design the BAL specifically to mimic the in vivo scenario. Such mathematical models enable theoretical hypothesis testing, will inform the design of in vitro experiments, and will enable both refinement and reduction of in vivo animal trials. In this way, development of novel mathematical modelling tools will help to focus and direct in vitro and in vivo research, and can be used as a framework for other areas of drug safety science