Cellular Agriculture Research Articles

Fiber-based Biomaterial Scaffolds for Cell Support towards the Production of Cultivated Meat

The in vitro production of animal-derived foods via cellular agriculture is emerging as a key solution to global food security challenges. Here, the potential for fiber-based scaffolds, including silk and cotton, in the cultivation of muscle cells for tissue formation was pursued. Mechanical properties and cytocompatibility with the mouse myoblast cell line C2C12 and immortalized bovine muscle satellite cells (iBSCs) were assessed, as well as pre-digestion options for the materials due to their resilience within the human digestive track. The fibers supported cell adhesion, proliferation, and guided muscle cell orientation, facilitating myotube formation per differentiation. A progressive increase in biomass was also documented. Interestingly, iBSC proliferation was enhanced with coatings of recombinant proteins while C2C12 cells showed minimal response. Thus, both cotton and silk yarns were suitable as fiber-based scaffolds towards cell supportive goals, suggesting an alternative path toward structured protein-rich foods via this initial stage of textile engineering for food. Biomass prediction models were generated, enabling forecasts of cell growth and maturation across various scaffold conditions and cell types. This capability enhances the precision of the cultivation process towards an engineering approach, building on the inherent benefits of hierarchical muscle tissue structure, but here via textile engineering with these initial muscle-coated edible fibers. Further, the approach offers to reduce costs by optimizing cultivation time and media needs. These approaches are part of a foundation for future scalable and sustainable cultivated meat production. Statement of SignificanceThis research investigates the use of one-dimensional fiber-based scaffolds for cultivated meat production, contributing to advancements in cellular agriculture. It introduces a method to measure changes in biomass and scaffold degradation throughout the cultivation process. Additionally, our development of biomass prediction models improves the precision and predictability of cultivated meat production. This research not only aids in scaling up cultivated meats but also enhances the use of textile engineering techniques in tissue engineering, paving the way for producing complex, three-dimensional meat structures more sustainably.

Morphological and Nutritional Analysis of White Shrimp, Litopenaeus setiferus, Muscle

In response to the growing seafood demand and the environmental impact of traditional aquaculture and fishing methods, this study explored the potential of cellular agriculture for sustainable seafood production. Focusing on shrimp, the most consumed seafood in the United States, our project aimed to establish a comprehensive database of the morphological and physicochemical properties of shrimp muscle. This database is intended to support the development of cell-based seafood by providing a benchmark for texture, color, and nutritional content. To do so, thirty large white shrimp (Litopenaeus setiferus) were analyzed through texture analysis, colorimetry, Hematoxylin and Eosin staining, and proximate analysis. The findings indicated consistent texture profile analysis parameters across the shrimp's surface and cross-section, while colorimetry values showed significant differences. H&E staining revealed similar cellular patterns, affirming the uniformity of shrimp texture. The proximate analysis offers a nutritional target for developing shrimp analogs. The study concludes that while shrimp texture is consistent, addressing color variation is crucial for the development of cell-based shrimp analogs. Future research should include characteristics of cooked shrimp and other seafood products to expand the database for cellular agriculture applications.

Proteomic study of secretomes from cellular agriculture for milk production

Cultivation of bovine mammary epithelial cells (bMECs) is regarded as a potential future source of milk constituents; however, there is a research gap concerning the investigation of compositional similarity between such secretomes and milk, and furthermore, how culture conditions can be optimized to make it a feasible strategy for potential production of milk constituents. To perform such an initial, detailed quantitative proteomic study of secretomes from in vitro grown bMECs, these cells were isolated from the mammary tissue of two lactating cows and cultured on Matrigel®-coated inserts in a trans-well system. Cells were treated with or without prolactin at the basolateral sides, and the secretomes together with media from the apical side were collected and subsequently compositionally characterized for their protein contents and label-free quantitative proteomics. Using sensitive nLC-timsTOF Pro MS/MS, more than 500 proteins were identified, though in very low total concentrations, across proteomes from the four secretomes collected from prolactin- or non-prolactin-treated cells. The proteins included many cellular proteins and characteristic milk proteins, but with caseins accounting for less than 1% of total protein abundance. Secretomes from prolactin-treated bMECs showed significant (P < 0.05) up-regulation of 351 different proteins, including milk proteins such as αS1-casein, κ-casein, β-lactoglobulin, lactotransferrin, and osteopontin, indicating a positive regulation of lactation-associated proteins with prolactin treatment. These findings elucidate a potential for future cellular agriculture using in vitro cultured bMECs for the production of milk constituents, together with challenges, such as upscale and sufficient lactogenesis.

Potato and dairy industry side streams as feedstock for fungal and plant cell cultures

Utilization of industrial side streams as nutrient source for cellular agriculture is a promising option to improve the sustainability of the production processes. The aim of the study was to evaluate usability of two liquid food industry side streams, potato cell fluid and acid whey, as nutrient source for production of food ingredients with plant cell cultures (arctic bramble (Rubus arcticus L.) and tobacco BY-2 (Nicotiana tabacum L.)) and filamentous fungi (Paecilomyces variotii, Rhizopus oligosporus and Trichoderma reesei). The side streams were found to contain various sugars and other potential nutrients suitable for the studied organisms. The side streams were used as a sole nutrient source (fungi) or as a replacement of selected nutrients in the growth media (fungi and plant cells) in flask scale cultivations. Acid whey was successfully used as a growth medium for filamentous fungi, but it inhibited plant cell growth presumably due to high organic acid content (14 g/l). Potato side stream was found suitable as a fungal and plant cell growth media supplement, and it was used together with glucose for filamentous fungi, or as a partial replacer of macronutrients for plant cells, in bioreactor cultivations yielding high fungal (up to 36 g/l dry weight) and good plant cell (9.5 g/l dry weight) biomass production. Analyses of the produced biomasses revealed good nutritional value in terms of amino acid (17–27 % of dry matter) and dietary fiber (23–30 % of dry matter) contents, giving good premises for utilization in human nutrition.

Would edible insects become part of our gastronomic quest?

Global food security status has been contested in recent times with claims of efficient food production by the proponents and severe to moderate food insecurity by the opponents. The current population growth prediction of 9.7 billion by 2050 requires additional food production due to the impact of climate change leading to diminished yield rates. Novel food sources such as cellular agriculture utilizing modern biotechnology are at various trial and commercialization stages. Old traditions of insects as a reliable food (entomophagy) source are gaining momentum in recent times. This article explores various aspects of entomophagy. The ecological footprint of entomophagy on its animal counterparts is presented. Since religious factors have a major impact on eating habits that privileges certain elements, perspectives from major religions on entomophagy have been discussed. The regulatory standpoint of Anglo-Saxon, Western and Eastern societies are given. Consumer knowledge and commercialization challenges are also discussed with concluding remarks.

Obtaining source material for cellular agriculture

Cellular Agriculture (CellAg) is an attractive concept for innovative technology with the intent to provide food and nutrition complementary to existing supply streams. The past decade has seen considerable progress in the field with advancement of cellular technology that delivers the initial building blocks for meaningful implementation. The availability of natural cell-based material that can serve as nutrient-filled source for human consumption at low cost is a critical challenge for the emerging cellular agriculture industry. Therefore, here the isolation of bovine myofibroblasts of the Black Angus breed has been pursued and accomplished together with its characterisation by using RNA sequencing and proteomics through western blotting.To transition CellAg from a concept to a game changing technology for the industry, multiple challenges need to be overcome. The field requires powerful initial material, i.e., dedicated cells that can proliferate and differentiate robustly at scale. The methodology described allows for the production of healthy cells, which have been unequivocally characterized as clonal representatives of a stable myofibroblast cell line using transcriptomics and proteomics validation. Stringent and rigorous live cell monitoring of a nascent cell line derived from healthy muscle tissue allowed for stable cell growth.In this research article, a simple and precise methodology is presented for creating a bovine myofibroblast cell line (Bov.mia). Our work puts forward a low-tech use of materials and expertise that is devoid of transgenic approaches, thus creating a reliable approach for lab-scale research.

Plant-based biomaterials as scaffolds for cellular agriculture

Plant-based biomaterials as scaffolds for cellular agriculture

Bovine Placentome-Derived Extracellular Matrix: A Sustainable 3D Scaffold for Cultivated Meat.

Cultivated meat, an advancement in cellular agriculture, holds promise in addressing environmental, ethical, and health challenges associated with traditional meat production. Utilizing tissue engineering principles, cultivated meat production employs biomaterials and technologies to create cell-based structures by introducing cells into a biocompatible scaffold, mimicking tissue organization. Among the cell sources used for producing muscle-like tissue for cultivated meats, primary adult stem cells like muscle satellite cells exhibit robust capabilities for proliferation and differentiation into myocytes, presenting a promising avenue for cultivated meat production. Evolutionarily optimized for growth in a 3D microenvironment, these cells benefit from the biochemical and biophysical cues provided by the extracellular matrix (ECM), regulating cell organization, interactions, and behavior. While plant protein-based scaffolds have been explored for their utilization for cultivated meat, they lack the biological cues for animal cells unless functionalized. Conversely, a decellularized bovine placental tissue ECM, processed from discarded birth tissue, achieves the biological functionalities of animal tissue ECM without harming animals. In this study, collagen and total ECM were prepared from decellularized bovine placental tissues. The collagen content was determined to be approximately 70% and 40% in isolated collagen and ECM, respectively. The resulting porous scaffolds, crosslinked through a dehydrothermal (DHT) crosslinking method without chemical crosslinking agents, supported the growth of bovine myoblasts. ECM scaffolds exhibited superior compatibility and stability compared to collagen scaffolds. In an attempt to make cultivate meat constructs, bovine myoblasts were cultured in steak-shaped ECM scaffolds for about 50 days. The resulting construct not only resembled muscle tissues but also displayed high cellularity with indications of myogenic differentiation. Furthermore, the meat constructs were cookable and able to sustain the grilling/frying. Our study is the first to utilize a unique bovine placentome-derived ECM scaffold to create a muscle tissue-like meat construct, demonstrating a promising and sustainable option for cultivated meat production.

Unveiling a Multiplexed Sensing System for Advanced Biosensing Applications with Thread-Based Electrochemical Sensors

The miniaturization and portability of electrochemical sensing devices play a crucial role in a wide range of applications, including medical diagnostics, environmental monitoring, and point-of-care testing. Integrating a pseudo-reference electrode addresses challenges linked to traditional counterparts, ensuring enhanced portability and miniaturization without performance compromise. Our research delves into the significance of this innovation, emphasizing its impact on signal stability, reproducibility, and overall sensor reliability. The tailored pseudo-reference for compact electrochemical devices contributes to a highly efficient, cost-effective, and user-friendly design, ideal for point-of-care diagnostics and field applications. This work showcases the design, implementation, and performance evaluation of a portable electrochemical sensing system with a pseudo-reference-integrated readout circuit, improving reliability under diverse operating conditions. In the proposed research, we present the design and implementation of a dual-channel configurable multiplexed sensing platform utilizing thread-based electrochemical sensors for continuous potentiometric and amperometric measurements.For the pH sensor, the fabrication process initiates with the cleaning (using isopropyl alcohol) and subsequent plasma treatment of polysorb sutures. The cleaned sutures undergo multiple coatings (4 cycles) with conductive carbon ink, followed by baking at 60°C for 30 to 35 minutes to ensure uniform conductivity. Carbon-coated (CC) sutures undergo electrochemical pre-treatment in a solution of 0.50 M H2SO4 and 0.10 M KCl, followed by electrodeposition with polyaniline (PANI) using potentiometric deposition at 0.80 V in the presence of 0.50 M aniline in a solution containing 1.0 M H2SO4. An insulating polymer layer of 5% polyurethane (PU) is applied at the sensor's center, leaving ends exposed for electrical connections and subsequent hydrogel coating for real sample analysis. The O2 sensor fabrication involves cleaning silver-coated threads, coating with dielectric ink for a sensing region, and UV curing. Two identical threads serve as the cathode and anode, with an intentionally longer anode region as a common reference for the pH sensor. This comprehensive approach contributes to developing a versatile and efficient sensing system for multiplexed measurements, showcasing potential applications in diverse environments.The measurement system includes a signal conditioning circuit (denoted as analog front-end or AFE in Fig. 1) comprising a trans-impedance amplifier (TIA) and an op-amp in the unity-gain configuration: the former is used to convert the current into voltage which originates from the oxygen sensor whereas the latter is used to read out the open-circuit voltage (OCV) for the pH sensor. The outputs of the TIA and the voltage buffer are sequentially selected using a 2:1 multiplexer with a control bit (SEL) before digitizing using an analog-to-digital converter (ADC). There is also a digital-to-analog converter (DAC) generating a bias voltage (VBIAS) of 0.55V for the oxygen sensor as the oxygen electrode requires a certain potential to maximize the dynamic range of the current measurement. The microcontroller handles digital signal processing, which can be connected to a computer for data storage and visualization. The operation of the sensor multiplexing system is validated in standard buffer solutions and gel mediums with different pH and O2 concentrations. The proposed setup demonstrated a sensitivity of ~65 mV/pH for the pH sensor (n=3), and 0.6621 nA/mgL-1 for the O2 sensor (n=3) in a gel medium with common ions and electrolytes. The sensor results were validated against commercial oxygen and pH probes for the same solutions. The developed sensing platform and sensor can precisely monitor and track changes in pH and O2 levels within less than 2 minutes for repeated cycles. This architecture has the potential for extending the low-power multiplexed sensing to multiple biomarkers with an increased number of sensors for ingestible devices, wearable sensing, and cellular agriculture applications. KEYWORDS: Multiplexed sensing, pseudo-reference, pH and oxygen monitoring, miniaturized circuit, thread-based electrochemical sensor Figure 1

Conditions for establishing fin primary cell cultures in a wide range of ray-finned fishes.

Ray-finned fishes (Actinopterygii) represent the most diverse vertebrate lineage that show extensive variations in physiology, ways of life, and adaptations to marine and freshwater environments, and several species have been established as biological research models. The in vitro culture of cells is fundamental for several fields of biological research, being an alternative for studies that use animals. Hundreds of fish cell lines have been established using specific methods for each cell type and species. Here is described a protocol which can be used commonly for obtaining cell cultures from the caudal fin of a wide range of ray-finned fishes including marine and freshwater species. Conditions for sample collection, microbial disinfection, tissue dissociation, plating and incubation, cryopreservation and thawing, and karyotyping are described in detail. Primary cell cultures were developed for 20 species grouped into 12 different orders. Eleven of these species have been cultivated in vitro for the first time. In the beginning, the fish cell cultures showed different capacities of proliferation among them; however throughout the passages, most cultures began to have a similar proliferation rate. Throughout the passages, it was noticed that cells similar to fibroblasts began to predominate. The great proliferative ability of these cultures reveals their potential to become cell lines. The culture of A. mexicanus, for example, has been proliferating for months and is already in its 65th passage. Moreover, these cell cultures showed conserved diploid chromosome numbers in comparison with in vivo descriptions which suggest these cultures have stable karyotypes. Therefore, these cultures have potential to be used in several fields, such as toxicology, cytogenetics, genomics, pathology, immunology, cellular agriculture, and conservation, and this method has the potential to be expanded to species not yet tested, as well as to other organs.

Prospects of Justice for Cellular Agriculture: A just Transition or Reinvesting in Unsustainability?

Transformation in food systems poses new opportunities for improving environmental sustainability and reducing the use of farmed animals. Discussions about transforming current food systems have been centered mostly on replacing animal source proteins with plant-based alternatives and about how to minimize food waste and loss. Products from cellular agriculture are part of a novel food transition and are presented as new, sustainable alternatives for animal source proteins. However, justice and equity narratives in food system transition discussions concerning cellular agriculture are rare. The aim of this study is to address how cellular agriculture may contribute to a just food system transition and to evaluate the prospects of such foods on this planet by reviewing narratives of cellular agriculture in 36 scientific articles. The data were analyzed using a justice transition framework. The results show that cellular agriculture has a potential to contribute to improving environmental sustainability if developers take justice into account as an important factor. Concerns are that cellular agriculture has the potential to be an exclusive food and may introduce regional variances exacerbating inequalities within the food system. We contribute to the discussion of just food system transitions by highlighting the importance of justice considerations in the context of cellular agriculture. Key aspects include the need for fair distribution along the value chain, global access to cellular agriculture benefits, and the recognition of social transformations in technological solutions. Additionally, transparent decision-making, open data access, and capacity building for stakeholders emerge as critical elements for fostering equitable and sustainable development in cellular agriculture.

Empirical economic analysis shows cost-effective continuous manufacturing of cultivated chicken using animal-free medium.

Cellular agriculture aims to meet the growing demand for animal products. However, current production technologies result in low yields, leading to economic projections that prohibit cultivated meat scalability. Here we use tangential flow filtration for continuous manufacturing of cultivated meat to produce biomass of up to 130 × 106 cells per ml, corresponding to yields of 43% w/v and multiple harvests for over 20 days. Continuous manufacturing was carried out in an animal-component-free culture medium for US$0.63 l-1 that supports the long-term, high density culture of chicken cells. Using this empirical data, we conducted a techno-economic analysis for a theoretical production facility of 50,000 l, showing that the cost of cultivated chicken can drop to within the range of organic chicken at US$6.2 lb-1 by using perfusion technology. Whereas other variables would also affect actual market prices, continuous manufacturing can offer cost reductions for scaling up cultivated meat production.

Chemical and sensory analyses of cultivated pork fat tissue as a flavor enhancer for meat alternatives

The emerging field of cellular agriculture has accelerated the development of cell-cultivated adipose tissue as an additive to enhance the flavor of alternative meat products. However, there has been limited research to evaluate the sensory profile of in vitro-grown tissues compared to conventionally obtained animal fat. This study aimed to investigate the aromatic characteristics of cell-cultivated fat tissue as a flavor enhancer for meat alternatives. Porcine dedifferentiated fat (PDFAT) cells were clonally isolated and differentiated into adipocytes. This cultured adipose tissue was then analyzed alongside native porcine fat using gas chromatography-mass spectrometry (GC/MS) coupled with descriptive sensory analysis by human consumers. This evaluation enabled quantitative and qualitative assessments of volatile compounds released during cooking for both in vitro and in vivo porcine fats. The volatile profiles generated during the cooking process and fatty aroma characteristics reported by sensory consumers were largely similar between the two fat sources, with some differences in select compounds and aroma attributes. Ultimately, the consumers found comparable overall liking scores reported between the conventional and cultured porcine fats. These findings provide valuable sensory evidence supporting the viability of cell-cultivated adipose tissue as a flavor component of meat alternatives, substituting for conventional animal fat.

Plant-based hydrolysates as building blocks for cellular agriculture

Cellular agriculture, an emerging technology, aims to produce animal-based products such as meat through scalable tissue culture methods. Traditional techniques rely on chemically undefined media using fetal bovine serum (FBS) or chemically defined media utilizing specific growth factors. To be a viable alternative to conventional meat production, cellular agriculture requires cost-effective materials with established supply chains for growth media.Here, we investigate hydrolysates from Kikuyu grass, Alfalfa grass, and cattle rearing pellets. We identified conditions that promote C2C12 myoblast cell growth in media containing 0.1% and 0% serum. These effects are more pronounced in combination with existing growth promoters such as insulin, transferrin, and selenium. Overall, the rearing pellet hydrolysates were most effective in promoting growth particularly when in combination with the growth promoters.Our findings suggest that leveraging these materials, along with known growth factors, can facilitate the development of improved, scalable, and commercially viable media for cellular agriculture.

Methods to Screen the Adhesion of Fish Cells on Plant-, Algal- and Fungal-Derived Biomaterials.

Cellular agriculture, an alternative and innovative approach to sustainable food production, has gained momentum in recent years. However, there is limited research into the production of cultivated seafood. Here, we investigated the ability of fish mackerel cells (Scomber scombrus) to adhere to plant, algal and fungal-based biomaterial scaffolds, aiming to optimize the cultivation of fish cells for use in cellular agriculture. A mackerel cell line was utilized, and metabolic assays and confocal imaging were utilized to track cell adhesion, growth, and differentiation on the different biomaterials. The mackerel cells adhered and grew on gelatin (positive control), zein, and soy proteins, as well as on alginate, chitosan, and cellulose polysaccharides. The highest adhesion and growth were on the zein and chitosan substrates, apart from the gelatin control. These findings provide a blueprint to enhance scaffold selection and design, contributing to the broader field of cellular agriculture through the development of scalable and eco-conscious solutions for meeting the growing global demand for seafood.

Identifying Nutritional Considerations to Inform Safety Assessments of Foods Derived From Cellular Agriculture: A Scoping Review Protocol

Identifying Nutritional Considerations to Inform Safety Assessments of Foods Derived From Cellular Agriculture: A Scoping Review Protocol

Isolation of a persistently quiescent muscle satellite cell population.

Although studies have identified characteristics of quiescent satellite cells (SCs), their isolation has been hampered by the fact that the isolation procedures result in the activation of these cells into their rapidly proliferating progeny (myoblasts). Thus, the use of myoblasts for therapeutic (regenerative medicine) or industrial applications (cellular agriculture) has been impeded by the limited proliferative and differentiative capacity of these myogenic progenitors. Here we identify a subpopulation of satellite cells isolated from mouse skeletal muscle using flow cytometry that is highly Pax7-positive, exhibit a very slow proliferation rate (7.7 ± 1.2 days/doubling), and are capable of being maintained in culture for at least 3 mo without a change in phenotype. These cells can be activated from quiescence using a p38 inhibitor or by exposure to freeze-thaw cycles. Once activated, these cells proliferate rapidly (22.7 ± 0.2 h/doubling), have reduced Pax7 expression (threefold decrease in Pax7 fluorescence vs. quiescence), and differentiate into myotubes with a high efficiency. Furthermore, these cells withstand freeze-thawing readily without a significant loss of viability (83.1 ± 2.1% live). The results presented here provide researchers with a method to isolate quiescent satellite cells, allowing for more detailed examinations of the factors affecting satellite cell quiescence/activation and providing a cell source that has a unique potential in the regenerative medicine and cellular agriculture fields.NEW & NOTEWORTHY We provide a method to isolate quiescent satellite cells from skeletal muscle. These cells are highly Pax7-positive, exhibit a very slow proliferation rate, and are capable of being maintained in culture for months without a change in phenotype. The use of these cells by muscle researchers will allow for more detailed examinations of the factors affecting satellite cell quiescence/activation and provide a novel cell source for the regenerative medicine and cellular agriculture fields.

Technological Aspects of Bridging the Gap Between Cell-Based Food and Conventional Meat

Cell-based food, including cultured meat, introduces an innovative complement to our dietary options, introducing cellular agriculture and tissue engineering on the meat market together with traditional livestock farming. Originating from medical tissue cultivation techniques, this approach is now tailored for food production, prioritizing cost-effectiveness, palatability, and resource efficiency. As technology strives to efficiently upscale production, consumer acceptance stands as a key factor in adopting this new protein source. This review explores advances in cultivating muscle and fat tissues in vitro, emphasizing the importance of achieving muscle maturity, innovating scaffolds, and optimizing media composition to closely replicate the qualities of meat. It also addresses quality assessments of cultured meat based on its texture, nutritional content, and flavor. A concise examination of consumer perceptions reveals that acceptance is influenced by a blend of cultural, psychological, and social factors, balancing the positive potential outlook on cultured meat’s benefits for society, the environment, and animal welfare against concerns about its unnaturalness, uncertainty, and safety. Demographic trends suggest higher acceptance among younger, well-educated and urban individuals, contrasting with reservations from those more familiar with the traditional meat sector. Addressing these varied viewpoints is essential for a better understanding of public acceptance if cultured meat is effectively introduced into our future food systems. As media interest in this alternative is still high, aligning technological developments with consumer expectations is crucial for the potential market introduction of cultured meat.

Lactogenic treatment effects on milk synthesis genes and protein secretion in cultured bovine mammary epithelial cells

In a cellular agriculture context, the possibility of producing milk components using tissue derived bovine mammary epithelial cells (MEC) is an upcoming strategy being considered. As an essential step, development of an in-vitro model for optimizing lactogenic activity of cultured MEC is needed. MECs were isolated from bovine mammary tissue and cultured in a trans-well system. Treatment with differentiation media (DM) was performed for 7-days. Conditioned media containing the cell secretions (the “secretomes”) were collected, and the trans-epithelial-resistance (TEER), measured. On day 7 the cells were lysed, and expression of lactation associated genes were examined using qPCR. Proteomic analysis of the secretomes was performed using timsToF Pro LC-MS/MS. DM treated cells had significantly higher TEER for several days but a general decreasing trend started on day 4. Expressions of CSN1S1, CSN2, CSN3, ELF5 and PRLR were significantly increased after DM treatment. DM significantly altered the secretome's protein compositions. Numerous proteins involved in lactogenic activity of the MEC were identified. However, the DM did not significantly impact their peak intensities. These findings shed light on areas requiring additional refinement of the in-vitro model to enhance the production of milk components. These aspects primarily comprise the DM composition and experimental length.

Optimizing interleukin-6 and 8 expression, clarification and purification in plant cell packs and plants for application in advanced therapy medicinal products and cellular agriculture

Healthcare and nutrition are facing a paradigm shift in light of advanced therapy medicinal products (ATMPs) and cellular agriculture options respectively. Both options heavily rely on some sort of animal cell culture, e.g. autologous stem cells. These cultures require various growth factors, such as interleukin-6 and 8 (IL-6/8), in a pure, safe and sustainable form that can be provided in a scalable manner. Plants seem well suited for this task because purification of small proteins can be readily achieved by membrane separation, human/animal pathogens do not replicate in plants and production can be scaled up using in-door farming or agricultural practices. Here, we illustrate this capacity by first optimizing the codon usage of IL-6/8 for translation in Nicotiana spp., as well as testing the effect of untranslated regions and product targeting to different sub-cellular compartments on expression in a high-throughput plant cell pack (PCP) assay. In the chloroplast, IL-6 accumulated up to 6.9±3.8 (SD, n=2) and 14.4±7.4 mg kg−1 (SD, n=5) were observed in case of IL-8. When transferring IL-8 expression into whole plants, accumulation was 12.3±1.5 mg kg−1 (SD, n=3). After extraction and clarification, IL-8 was purified using a two-stage process consisting of an ultrafiltration/diafiltration step with 100 kDa and 10 kDa cut off membranes followed by an IMAC polishing step. The purity, yield and recovery were 97.8%, 6.6 mg kg−1 and 38%, respectively. We evaluated the ability of the proposed purification process to remove endotoxins to ensure the compatibility of plant-made growth factors with cell culture.