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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.

Mammary epithelial cells (MEC) are responsible for milk synthesis in the bovine mammary gland during lactation, and could thereby potentially be used for in-vitro milk components production. Development of such a model, however, requires the MECs to preserve their lactogenic traits following in-vitro growth. Potential impact of growing MEC in the lab following their isolation was studied in relation to their population homogeneity and milk synthesis related gene expressions. Upon isolation, flow cytometry results showed that > 30 % of the obtained cells were positive for cytokeratin 18 (CK18), a marker for the MEC. This proportion increased to 95 % MEC after the first passage. Cell functionality was examined by comparing the expressions of milk synthesis related genes at passage 2 (P2) to passage 6 (P6) following treatment with lactogenic agents. CSN1S1 was found to be expressed significantly higher at P2 compared to P6. In addition, CSN2, CSN3 and PRLR tended to have lower expressions in P6 compared to P2. DGAT1, STAT5A, AKT1, and RPS6KB1 were expressed comparably in both passages. The expressions of LALBA and CSN1S2 were not detectable in any of the samples. Therefore, despite having high levels of CK18 positive cells at later passages, in line with other studies, further passaging affected the expressions of some of the genes related to milk synthesis. A reduced gene expression capacity in the propagated MEC may limit their potential for in-vitro milk components production.

Reducing cost of growth factors in fetal bovine serum free cell culture media is critical for cultivated meat production. In this issue, Stout et al. demonstrate how to overexpress the growth factors fibroblast growth factor 2 and its amplifier RasG12V in immortalized bovine satellite cells (iBSCs).

Colloidal delivery systems improve the oral bioavailability of lipid-soluble bioactives, providing protection throughout the gastrointestinal tract. In this study, the fate of a model bioactive, curcumin, was followed during in vitro digestion and absorption, using state of the art INFOGEST in vitro models. Curcumin was loaded in zein nanoparticles, prepared by antisolvent precipitation, with or without sodium caseinate (Cur-Z-C and Cur-Z, respectively). The bioefficacy of the digesta were evaluated using Caco-2 cells. In addition, the absorption of curcumin was evaluated using a co-culture of Caco-2/HT29-MTX cells, which are mucus producing. Ultra-high-performance quadrupole time-of-flight mass spectrometry and cellular bioassays were utilized to assess the presence of bioaccessible material, and any residual curcumin after absorption. Zein nanoparticles were fully digested during gastrointestinal transit. A comparison between aqueous curcumin and digested Cur-Z and Cur-Z-C showed that Cur-Z-C decreased the proliferation of Caco-2 cells by ∼55%. This finding points out the importance of matrix effect on the efficacy of delivery systems.

Summary: Culturing eukaryotic cells has widespread applications in research and industry, including the emerging field of cell-cultured meat production colloquially referred to as “cellular agriculture”. These applications are often restricted by the high cost of growth medium necessary for cell growth. Mitogenic protein growth factors (GFs) are essential components of growth medium and account for upwards of 90% of the total costs. Here, we present a set of expression constructs and a simplified protocol for recombinant production of functionally active GFs, including FGF2, IGF1, PDGF-BB, and TGF-β1 in Escherichia coli. Using this E. coli expression system, we produced soluble GF orthologs from species including bovine, chicken, and salmon. Bioactivity analysis revealed orthologs with improved performance compared to commercially available alternatives. We estimated that the production cost of GFs using our methodology will significantly reduce the cost of cell culture medium, facilitating low-cost protocols tailored for cultured meat production and tissue engineering.

The hepatic cytochrome p450’s (CYP) are of major importance for the metabolism of xenobiotics and knowledge about their regulation is crucial. This knowledge often originates from cell models; primary human hepatocytes (PHH) being the gold standard. However, due to limited availability of high-quality human donor organs, basic knowledge on alternative models are needed. Primary porcine hepatocytes (PPH) have been suggested as an alternative to PHH. Unfortunately, data comparing the response in gene-transcription to standard CYP inducers between PHH and PPH are missing. In the present study we, cultured PHH and PPH under the same conditions, treated them with standard inducers of the CYP1-3 and determined the response in gene and protein expression. The results demonstrated that in both species TCDD and omeprazole caused an increase in CYP1A/B expression. In PPH, CITCO increased the content of CYP1A/B. For the CYP2B/C/D’s, phenobarbital and rifampicin caused increases in expression. For the CYP2D’s, TCDD and omeprazole caused increased gene expression in PPH, which were not the case for PHH. Both phenobarbital, rifampicin and omeprazole increased CYP3A expression in PHH and PPH. Moreover, TCDD increased the gene expression of CYP3A in PPH; this was not the case for PHH. Multivariate data analysis found no difference in gene expression between PHH and PPH for phenobarbital, rifampicin and CITCO. However, differential clustering was observed for TCDD and omeprazole. In conclusion, despite model specificity, there are a high number of similar responses, and experiments investigating mRNA regulation made in PPH permits for a reliable translation into human setting.

The occurrence of wooden breast (WB) in broiler production is increasing, but onset of its development is only described in part. In this study, we determined the regulation of marker genes related to oxidative stress in Ross308 broilers categorized as no-, mild- or severe-WB, on days 21 and 30 of production. The biochemical parameters, lactate dehydrogenase and pro- and macro-glycogen, were also determined. On day 21, breast meat from birds affected severely by WB had increased mRNA abundances of heat-shock protein 70, heme-oxygenase 1, cyclooxygenase 2, tumor necrosis factor 1, and hypoxia inducible factors as well as higher pH and lower dry matter contents. On day 30, breast meat from both mild and severely affected birds had increased mRNA for heme oxygenase 1, lactate dehydrogenase, and hypoxia inducible factor. Moreover, pro- and micro-glycogen, as well as the total pool of glycogen, were decreased compared with the non-WB birds. In conclusion, this study indicates oxidative stress, inflammation and hypoxic conditions in WB.

Red meat has been associated with an increased cardiovascular disease (CVD) risk, possibly through gut microbial-derived trimethylamine-N-oxide (TMAO). However, previous reports are conflicting, and influences from the background diet may modulate the impact of meat consumption. This study investigated the effect of red and white meat intake combined with two different background diets on urinary TMAO concentration and its association with the colon microbiome in addition to apparent hepatic TMAO-related activity. For 4 weeks, 32 pigs were fed chicken or red and processed meat combined with a prudent or western background diet. 1H NMR-based metabolomics analysis was conducted on urine samples and hepatic Mrna expression of TMAO-related genes determined. Lower urinary TMAO concentrations were observed after intake of red and processed meat when consumed with a prudent compared to a western background diet. In addition, correlation analyses between urinary TMAO concentrations and relative abundance of colon bacterial groups suggested an association between TMAO and specific bacterial taxa. Diet did not affect the hepatic Mrna expression of genes related to TMAO formation. The results suggest that meat-induced TMAO formation is regulated by mechanisms other than alterations at the hepatic gene expression level, possibly involving modulations of the gut microbiota.

Cytochrome P450 (CYP450) is the major family of enzymes involved in the metabolism of several xenobiotic and endogenous compounds. Among substrates for CYP450 is the tryptophan metabolite skatole (3-methylindole), one of the major contributors to the off-odour associated with boar-tainted meat. The accumulation of skatole in pigs is highly dependent on the hepatic clearance by CYP450s. In recent years, the porcine CYP450 has attracted attention both in relation to meat quality and as a potential model for human CYP450. The molecular regulation of CYP450 mRNA expression is controlled by several nuclear receptors and transcription factors that are targets for numerous endogenously and exogenously produced agonists and antagonists. Moreover, CYP450 expression and activity are affected by factors such as age, gender and feeding. The regulation of porcine CYP450 has been suggested to have more similarities with human CYP450 than other animal models, including rodents. This article reviews the available data on porcine hepatic CYP450s and its implications for boar taint.

Skatole (3-methylindole) is a product of bacterial fermentation of tryptophan in the intestine. A significant amount of skatole can also be inhaled during cigarette smoking. Skatole is a pulmonary toxin that induces the expression of aryl hydrocarbon receptor (AhR) regulated genes, such as cytochrome P450 1A1 (CYP1A1), in human bronchial cells. The liver has a high metabolic capacity for skatole and is the first organ encountered by the absorbed skatole; however, the effect of skatole in the liver is unknown. Therefore, we investigated the impact of skatole on hepatic AhR activity and AhR-regulated gene expression. Using reporter gene assays, we showed that skatole activates AhR and that this is accompanied by an increase of CYP1A1, CYP1A2 and CYP1B1 expression in HepG2-C3 and primary human hepatocytes. Specific AhR antagonists and siRNA-mediated AhR silencing demonstrated that skatole-induced CYP1A1 expression is dependent on AhR activation. The effect of skatole was reduced by blocking intrinsic cytochrome P450 activity and indole-3-carbinole, a known skatole metabolite, was a more potent inducer than skatole. Finally, skatole could reduce TCDD-induced CYP1A1 expression, suggesting that skatole is a partial AhR agonist. In conclusion, our findings suggest that skatole and its metabolites affect liver homeostasis by modulating the AhR pathway.