Human cell lines, first cultured in the 1950s 1 , are indispensable in biomedical research. Today, a wide range of cell types are available, and sophisticated advanced 'omics' and visualization techniques allow for the routine assessment of cell identity and cellular responses 2 . However, the culture methods have remained relatively unchanged. Major advances in culture systems were made over three decades ago 3,4 , yet the old standard approach of batch cell culturethe culture of cells either in suspension or as adherent monolayers of cells in standard media [5][6][7] remains the predominant method in biomedical research.Culture media provides crucial nutrients, signalling molecules (such as growth factors), and suitable osmotic conditions. The gaseous and thermal environments of cell cultures are typically controlled by the incubator. The initial media conditions are generally stabilized by adjusting them to 18.6% O2 and a standard pH of 7.4, and this adjustment is achieved by adding a given amount of HCO3 − salt (a base), and by enriching the media with CO2 to a given percentage in the air (usually 5% or 10%). However, cell metabolism involves the exchange of gasesspecifically the release of CO2 and the consumption of O2and this can affect cellular growth via the alteration of, for example, the pH and the level of dissolved O2 (dO2) in the cellular microenvironment 8 . In theory, the equilibration of the medium with the gaseous and thermal environments of the incubator provides a way to reliably mimic O2, CO2 and HCO3 − homeostasis in metazoan body fluids. Yet this doesn't take into account the fact that homeostasis in a living mammal is supported by the active exchange of gases with the atmosphere. The absence of such active gas exchange in cell cultures suggests that, over time, cellular metabolic activities might acidify and deoxygenate the cellular microenvironment 8-10 , if intermittent monitoring and (when necessary) corrective action are not carried out.To mimic a physiological environment when using cell cultures, careful control over environmental factors (such as pH, CO2 and O2) is typically needed, in particular because even small deviations of environmental parameters from physiological levels may impair cellular function. For instance, in human blood, pH values below 7.2 (acidaemic conditions) and above 7.44 (alkalaemic conditions) can be fatal [11][12][13] . In cell cultures, the optimal growth of normal cells (that is, non-cancerous cells and non-transformed cells) occurs within a specific alkaline pH range, whereas cancer cells grow in a broader pH range that is shifted towards acidic values [14][15][16][17][18] . Cells have evolved mechanisms, including the use of Na + /H + antiporters or histone deacetylation, that restore the alkaline pH of the cytoplasm when the extracellular pH deviates from physiological levels [19][20][21][22][23][24][25] . However, such regulatory mechanisms require cellular energy, and changes in the acetylation state of chromatin can alter gene transcription and reduc...

This study assesses for the first time the ingestion of microplastics by giant clams and evaluates their importance as a sink for this pollutant. A total of 24 individuals of two size classes were collected from the Red Sea and then exposed for 12 days to 4 concentrations of polyethylene microbeads ranging from 53 to 500 µm. Experiments revealed that clams actively take up microplastic from the water column and the average of beads retained inside the animal was ~ 7.55 ± 1.89 beads individual -1 day -1 (5.76 ± 1.16 MPs / g dw). However, the digestive tract itself cannot be considered the only sink of microbeads in Tridacnids. Indeed, shells play a key role as well. The abundance of microplastic adhering to the shells, which was estimated directly, was positively correlated to the concentration of beads found in the surrounding seawater.Therefore, clams' shells contribute to the removal of 66.03 ± 2.50 % of the microplastic present in the water column. Furthermore, stress responses to the exposure to polyehylene were investigated. Gross Primary Production:Respiration (GPP:R) ratio decreased throughout of the experiment, but no significant difference was found between treatments and controls.

The characterization of the internal microenvironment of symbiotic marine invertebrates is essential for a better understanding of the symbiosis dynamics. Microalgal symbionts (of the family: Symbiodiniaceae) influence diel fluctuations of in host O2 and pH conditions through their metabolic activities (i.e., photosynthesis and respiration). These variations may play an important role in driving oxygen budgets and energy demands of the holobiont and its responses to climate change. In situ measurements using microsensors were used to resolve the O2 and pH diel fluctuations in the oral arms of non-calcifying cnidarian model species Cassiopea sp. (the “upside-down jellyfish”), which has an obligatory association with Symbiodiniaceae. Before sunrise, the internal O2 and pH levels were substantially lower than those in ambient seawater conditions (minimum average levels: 61.92 ± 5.06 1SE μmol O2 L–1 and 7.93 ± 0.02 1SE pH units, respectively), indicating that conditions within Cassiopea’s oral arms were acidified and hypoxic relative to the surrounding seawater. Measurements performed during the afternoon revealed hyperoxia (maximum average levels: 546.22 ± 16.45 1SE μmol O2 L–1) and internal pH similar to ambient levels (8.61 ± 0.02 1SE pH units). The calculated gross photosynthetic rates of Cassiopea sp. were 0.04 ± 0.013 1SE nmol cm–2 s–1 in individuals collected at night and 0.08 ± 0.02 1SE nmol cm–2 s–1 in individuals collected during the afternoon.

Mammalian cell cultures are a keystone resource in biomedical research, but the results of published experiments often suffer from reproducibility challenges. This has led to a focus on the influence of cell culture conditions on cellular responses and reproducibility of experimental findings. Here, we perform frequent in situ monitoring of dissolved O2 and CO2 with optical sensor spots and contemporaneous evaluation of cell proliferation and medium pH in standard batch cultures of three widely used human somatic and pluripotent stem cell lines. We collate data from the literature to demonstrate that standard cell cultures consistently exhibit environmental instability, indicating that this may be a pervasive issue affecting experimental findings. Our results show that in vitro cell cultures consistently undergo large departures of environmental parameters during standard batch culture. These findings should catalyze further efforts to increase the relevance of experimental results to the in vivo physiology and enhance reproducibility.

In vitro models are emerging tools for reducing reliance on traditional toxicity tests, especially in areas where information is sparse. For studies of fish, this is especially important for extrahepatic organs, such as the intestine, which, until recently, have been largely overlooked in favour of the liver or gill. Considering the importance of dietary uptake of contaminants, the rainbow trout (Oncorhynchus mykiss) intestine-derived cell line RTgutGC was cultured, to test its suitability as a high-throughput in vitro model. Benzo[a]pyrene (B[a]P) is an important contaminant and a model polycyclic aromatic hydrocarbon (PAH). Over 48 h exposure, a range of endpoints and xenobiotic metabolism rates were examined at three different pH levels indicative of the in vitro (pH 7.5) and in vivo mid-gut (pH 7.7) and hind-gut (pH 7.4) regions as a function of time. These endpoints included (i) cell viability: acid phosphatase (APH) and lactate dehydrogenase (LDH) assays; (ii) glucose uptake; (iii) cytochrome P450 enzyme activity: 7-ethoxyresoorufin-O-deethylase (EROD) assay; (iv) glutathione transferase (GST) activity; (v) genotoxic damage determined using the comet assay. Absence of cell viability loss, in parallel with decrease in the parent compound (B[a]P) in the medium and its subsequent increase in the cells suggested active sequestration, biotransformation, and removal of this representative PAH. With respect to genotoxic response, significant differences were observed at both the sampling times and the two highest concentrations of B[a]P. No significant differences were observed for the different pH conditions. Overall, this in vitro xenobiotic metabolism system appears to be a robust model, providing a basis for further development to evaluate metabolic and toxicological potential of contaminants without use of animals.