Understanding PFAS toxicity through cell culture metabolomics: Current applications and future perspectives.

Per- and polyfluoroalkyl substances (PFAS), ubiquitous environmental contaminants, pose significant challenges to ecosystems and human health. While cell cultures have emerged as new approach methodologies (NAMs) in ecotoxicity research, metabolomics is an emerging technique used to characterize the small-molecule metabolites present in cells and to understand their role in various biological processes. Integration of metabolomics with cell cultures, known as cell culture metabolomics, provides a novel and robust tool to unravel the complex molecular responses induced by PFAS exposure. In vitro testing also reduces reliance on animal testing, aligning with ethical and regulatory imperatives. The current review summarizes key findings from recent studies utilizing cell culture metabolomics to investigate PFAS toxicity, highlighting alterations in metabolic pathways, biomarker identification, and the potential linkages between metabolic perturbations. Additionally, the paper discusses different types of cell cultures and metabolomics methods used for studies of environmental contaminants and particularly PFAS. Future perspectives on the combination of metabolomics with other advanced technologies, such as single-cell metabolomics (SCM), imaging mass spectrometry (IMS), extracellular flux analysis (EFA), and multi-omics are also explored, which offers a holistic understanding of environmental contaminants. The synthesis of current knowledge and identification of research gaps provide a foundation for future investigations that aim to elucidate the complexities of PFAS-induced cellular responses and contribute to the development of effective strategies for mitigating their adverse effects on human health.

Fluid-Dynamic Culture of Tumour and Immune Cells for More Predictive Infiltration Studies and Immunotherapy Drug Screening.

Immunotherapies represent one of the current most promising challenges in cancer treatment. They are based on the boost of natural immune responses, aimed at cancer eradication. However, the success of immunotherapeutic approaches strictly depends on the interaction between immune cells and cancer cells. Preclinical drug tests currently available are poor in fully predicting the actual safety and efficacy of immunotherapeutic treatments under development. Indeed, conventional 2D cell culture underrepresents the complexity of the tumour microenvironment, while in vivo animal models lack in mimicking the human immune cell responses. In this context, predictability, reliability, and complete immune compatibility still represent challenges to overcome. For this aim, novel 3D, fully humanized in vitro cancer tissue models have been recently optimized by adopting emerging technologies, such as organ-on-chips (OOC) and 3D cancer cell-laden hydrogels. In particular, a novel multi-in vitro organ (MIVO) OOC platform has been recently adopted to culture 3D clinically relevant size cancer tissues under proper physiological culture conditions to investigate anti-cancer treatments and immune-tumour cell crosstalk.The proposed immune-tumour OOC-based model offers a potential tool for accurately modelling human immune-related diseases and effectively assessing immunotherapy efficacy, finally offering promising experimental approaches for personalized medicine.

Phenolic Determination in Proembryogenic Cell Complexes of Buckwheat Morphogenic Cell Culture with Osmium Tetroxide, Toluidine Blue O Dye, and Iron Chloride.

The study of the localization of secondary metabolites in both plants and the cell cultures on the intravital sections is hampered by the difficulty of obtaining thin, correctly oriented sections. Techniques for fixing tissues in resins allow these difficulties to be overcome. Properly selected tissue fixation techniques allow using different dyes to identify the compound of interest. In addition, some components of tissue fixation can act as fixatives and as a dye for identifying secondary metabolites. For example, osmium tetroxide, which fixes lipids in tissues, stains phenolic compounds black. This paper describes methods for the detection of phenolic compounds in morphogenic callus culture of buckwheat using osmium tetroxide, Toluidine Blue O dye, and ferric chloride as dyes in epoxy resin-embedded cell culture with double fixation of the material and when material fixed in Karnovsky's fixative.

Integrated Omics Approach: Revealing the Mechanism of Auxenochlorella pyrenoidosa Protein Extract Replacing Fetal Bovine Serum for Fish Muscle Cell Culture.

The process of producing cell-cultured meat involves utilizing a significant amount of culture medium, including fetal bovine serum (FBS), which represents a considerable portion of production expense while also raising environmental and safety concerns. This study demonstrated that supplementation with Auxenochlorella pyrenoidosa protein extract (APE) under low-serum conditions substantially increased Carassius auratus muscle (CAM) cell proliferation and heightened the expression of Myf5 compared to the absence of APE. An integrated intracellular metabolomics and proteomics analysis revealed a total of 13 and 67 differentially expressed metabolites and proteins, respectively, after supplementation with APE in the medium containing 5%FBS, modulating specific metabolism and signaling pathways, which explained the application of APE for passage cell culture under low-serum conditions. Further analysis revealed that the bioactive factors in the APE were protein components. Moreover, CAM cells cultured in reconstructed serum-free media containing APE, l-ascorbic acid, insulin, transferrin, selenium, and ethanolamine exhibited significantly accelerated growth in a scale-up culture. These findings suggest a promising alternative to FBS for fish muscle cell culture that can help reduce production costs and environmental impact in the production of cultured meat.

Regulation of STAT1 and STAT4 Expression by Growth Factor and Interferon Supplementation in Sjögren's Syndrome Cell Culture Models.

Our goal was to investigate the effects of epidermal growth factor (EGF) and interferons (IFNs) on signal transducer and activator of transcription STAT1 and STAT4 mRNA and active phosphorylated protein expression in Sjögren's syndrome cell culture models. iSGECs (immortalized salivary gland epithelial cells) and A253 cells were treated with EGF, IFN-alpha, -beta, -gamma, or mitogen-activated protein kinase p38 alpha (p38-MAPK) inhibitor for 0-24-48-72 h. STAT1 and STAT4 mRNA expression was quantified by qRT-PCR. Untreated and treated cells were compared using the delta-delta-CT method based on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) normalized relative fold changes. phospho-tyrosine-701-STAT1 and phospho-serine-721-STAT4 were detected by Western blot analysis. STAT4 mRNA expression decreased 48 h after EGF treatment in A253 cells, immortalized salivary gland epithelial cells iSGECs nSS2 (sicca patient origin), and iSGECs pSS1 (anti-SSA negative Sjögren's Syndrome patient origin). EGF and p38-MAPK inhibitor decreased A253 STAT4 mRNA levels. EGF combined with IFN-gamma increased phospho-STAT4 and phospho-STAT1 after 72 h in all cell lines, suggesting additive effects for phospho-STAT4 and a major effect from IFN-gamma for phospho-STAT1. pSS1 and nSS2 cells responded differently to type I and type II interferons, confirming unique functional characteristics between iSGEC cell lines. EGF/Interferon related pathways might be targeted to regulate STAT1 and STAT4 expression in salivary gland epithelial cells. Further investigation is required learn how to better target the Janus kinases/signal transducer and activator of transcription proteins (JAK/STAT) pathway-mediated inflammatory response in Sjögren's syndrome.

Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture.

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R's (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.

Cell culture models for assessing the effects of bioactive compounds in common buckwheat (Fagopyrum esculentum): a systematic review.

Common buckwheat (CBW) is grown and consumed worldwide. In addition to its already established reputation as an excellent source of nutrients, CBW is gaining popularity as a possible component of functional foods. Whereas human studies remain the gold standard for evaluating the relationship between nutrition and health, the development of reliable in vitro or ex vivo models has made it possible to investigate the cellular and molecular mechanisms of CBW effects on human health. Herein is a systematic review of studies on the biological effect of CBW supplementation, as assessed on various types of cellular models. Although the studies reported here have been conducted in very different experimental conditions, the overall effects of CBW supplementation were found to involve a decrease in cytokine secretion and oxidation products, related mainly to CBW polyphenols and protein or peptide fractions. These chemical species also appeared to be involved in the modulation of cell signaling and hormone secretion. Although further studies are undoubtedly necessary, as is their extension to in vivo systems, these reports suggest that CBW-based foods could be relevant to maintaining and/or improving human health and the quality of life.

Innovative explorations: unveiling the potential of organoids for investigating environmental pollutant exposure.

As the economy rapidly develops, chemicals are widely produced and used. This has exacerbated the problems associated with environmental pollution, raising the need for efficient toxicological evaluation techniques to investigate the toxic effects and mechanisms of toxicity of environmental pollutants. The progress in the techniques of cell culture in three dimensions has resulted in the creation of models that are more relevant in terms of biology and physiology. This enables researchers to study organ development, toxicology, and drug screening. Adult stem cells (ASCs) and induced pluripotent stem cells (iPSCs) can be obtained from various mammalian tissues, including cancerous and healthy tissues. Such stem cells exhibit a significant level of tissue memory and ability to self-assemble. When cultivated in 3D in vitro environments, the resulting organoids demonstrate a remarkable capacity to recapitulate the cellular composition and function of organs in vivo. Recently, many tumors' tissue-derived organoids have been widely used in research on tumor pathogenesis, drug development, precision medicine, and other fields, including those derived from colon cancer, cholangiocarcinoma, liver cancer, and gastric cancer. However, the application of organoid models for evaluating the toxicity of environmental pollutants is still in its infancy. This review introduces the characteristics of the toxicity responses of organoid models upon exposure to pollutants from the perspectives of organoid characteristics, tissue types, and their applications in toxicology; discusses the feasibility of using organoid models in evaluating the toxicity of pollutants; and provides a reference for future toxicological studies on environmental pollutants based on organoid models.

Salacia spp.: recent insights on biotechnological interventions and future perspectives.

The plants of the genus Salacia L. are the storehouse of several bioactive compounds, and are involved in treating human diseases and disorders. Hitherto, a number of reports have been published on in vitro biotechnology as well as microbial involvement in the improvement of Salacia spp. The present review provides comprehensive insights into biotechnological interventions such as tissue culture for plant propagation, in vitro cultures, and endophytic microbes for up-scaling the secondary metabolites and biological potential of Salacia spp. Other biotechnological interventions such as molecular markers and bio-nanomaterials for up-grading the prospective of Salacia spp. are also considered. The in vitro biotechnology of Salacia spp. is largely focused on plant regeneration, callus culture, cell suspension culture, somatic embryogenesis, and subsequent ex vitro establishment of the in vitro-raised plantlets. The compiled information on tissue cultural strategies, involvement of endophytes, molecular markers, and nanomaterials will assist the advanced research related to in vitro manipulation, domestication, and commercial cultivation of elite clones of Salacia spp. Moreover, the genetic diversity and other molecular-marker based assessments will aid in designing conservation policies as well as support upgrading and breeding initiatives for Salacia spp. KEY POINTS: • Salacia spp. plays a multifaceted role in human health and disease management. • Critical and updated assessment of tissue culture, endophytic microbes, metabolites, molecular markers, and bio-nanomaterials of Salacia spp. • Key shortcomings and future research directions for Salacia biotechnology.

The potential of Rhodobacter sphaeroides extract as an alternative supplement for cell culture systems.

It is essential to identify suitable supplements that enhance cell growth, viability, and functional development in cell culture systems. The use of fetal bovine serum (FBS) has been common, but it has limitations, such as batch-to-batch variability, ethical concerns, and risks of environmental contamination. In this study, we explore the potential of Rhodobacter sphaeroides extract, derived from a probiotic photosynthetic bacterium, as an alternative supplement. Our results demonstrate that the extract from R. sphaeroides significantly improves various aspects of cell behavior compared to serum-free conditions. It enhances cell growth and viability to a greater extent than FBS supplementation. Additionally, the extract alleviates oxidative stress by reducing intracellular levels of reactive oxygen species and stimulates lysosomal activity, contributing to cellular processes. The presence of abundant amino acids, glycine and arginine, in the extract may play a role in promoting cell growth. These findings emphasize the potential of R. sphaeroides extract as a valuable supplement for cell culture, offering advantages over the use of FBS.IMPORTANCEThe choice of supplements for cell culture is crucial in biomedical research, but the widely used fetal bovine serum (FBS) has limitations in terms of variability, ethics, and environmental risks. This study explores the potential of an extract from Rhodobacter sphaeroides, a probiotic bacterium, as an alternative supplement. The findings reveal that the R. sphaeroides extract surpasses FBS in enhancing cell growth, viability, and functionality. It also mitigates oxidative stress and stimulates lysosomal activity, critical for cellular health. The extract's abundance of glycine and arginine, amino acids with known growth-promoting effects, further highlights its potential. By providing a viable substitute for FBS, the R. sphaeroides extract addresses the need for consistent, ethical, and environmentally friendly cell culture supplements. This research paves the way for sustainable and reliable cell culture systems, revolutionizing biomedical research and applications in drug development and regenerative medicine.

Highly Efficient Verbascoside Production from Olive (Olea europea L. var. Cellina di Nardò) In Vitro Cell Cultures.

Olive (Olea europea L.) is one of the oldest and most important fruit tree species cultivated in the Mediterranean region. Various plant tissues, drupes, and olive oil contain several phenolics (including verbascoside, although it is present in the plant at a low level) that are well-known for their highly beneficial effects on human health. An in vitro olive cell suspension culture (cultivar Cellina di Nardò, "CdN") was established, characterized for its growth and morphological features. Furthermore, a vital and relatively uniform population of protoplasts was generated from the olive suspension culture to investigate their cellular characteristics during growth. The polyphenolic extract of the in vitro "CdN" olive cells contained almost exclusively verbascoside, as revealed by the UPLC-ESI-MS analysis. The content of verbascoside reached up to 100 mg/g DW, with an average production rate of approximately 50 mg/g DW over one year of culture. This level of production has not been previously reported in a limited number of previous studies. This remarkable production of verbascoside was associated with an exceptionally high antioxidant capacity. The high level of verbascoside production and purity of the extract make this system a promising tool for secondary metabolite production.

Single molecule real-time sequencing data sets of Hypericum perforatum L. plantlets and cell suspension cultures.

Hypericum is a large genus that includes more than 500 species of pharmacological, ecological and conservation value. Although latest advances in sequencing technologies were extremely exploited for generating and assembling genomes of many living organisms, annotated whole genome sequence data is not publicly available for any of the Hypericum species so far. Bioavailability of secondary metabolites varies for different tissues and the data derived from different cultures will be a valuable tool for comparative studies. Here, we report the single molecule real-time sequencing (SMRT) data sets of Hypericum perforatum L. plantlets and cell suspension cultures for the first time. Sequencing data from cell suspension cultures yielded more than 33,000 high-quality transcripts from 20 Gb of raw data, while more than 55,000 high-quality transcripts were obtained from 35 Gb of raw data from plantlets. This dataset is a valuable tool for comparative transcriptomic analysis and will help to understand the unknown biosynthetic pathways of high medicinal value in the Hypericum genus.

Suspension culture of stem cells established of Calendula officinalis L.

Plant stem cell cultures have so far been established in only a few plant species using cambial meristematic cells. The presence of stem cells or stem cell-like cells in other organs and tissues of the plant body, as well as the possibility of de novo generation of meristematic cells from differentiated cells, allow to consider the establishment of stem cell cultures in a broader range of species. This study aimed to establish a stem cell culture of the medicinal plant Calendula officinalis L. Callus tissues were induced from leaf and root explants, and already at this stage, stem and dedifferentiated cells could be identified. The cell suspension cultures established both from the root- and leaf-derived calli contained a high proportion of stem cells (92-93% and 72-73%, respectively). The most effective combination of growth regulators for the development of stem cells in calli as well as cell cultures was 1.0 mg/L 2,4-D and 0.5 mg/L BAP. The highest amount of stem cells (5.60-5.72 × 105) was in cell suspension derived from the roots. An effective protocol for the establishment of marigold stem cell suspension culture was developed. The ratio of root-derived stem cells against dedifferentiated cells exceeded 90%.

Advances in screening hyperthermic nanomedicines in 3D tumor models.

Hyperthermic nanomedicines are particularly relevant for tackling human cancer, providing a valuable alternative to conventional therapeutics. The early-stage preclinical performance evaluation of such anti-cancer treatments is conventionally performed in flat 2D cell cultures that do not mimic the volumetric heat transfer occurring in human tumors. Recently, improvements in bioengineered 3D in vitro models have unlocked the opportunity to recapitulate major tumor microenvironment hallmarks and generate highly informative readouts that can contribute to accelerating the discovery and validation of efficient hyperthermic treatments. Leveraging on this, herein we aim to showcase the potential of engineered physiomimetic 3D tumor models for evaluating the preclinical efficacy of hyperthermic nanomedicines, featuring the main advantages and design considerations under diverse testing scenarios. The most recent applications of 3D tumor models for screening photo- and/or magnetic nanomedicines will be discussed, either as standalone systems or in combinatorial approaches with other anti-cancer therapeutics. We envision that breakthroughs toward developing multi-functional 3D platforms for hyperthermia onset and follow-up will contribute to a more expedited discovery of top-performing hyperthermic therapies in a preclinical setting before their in vivo screening.

Enhancing Osteoblastic Cell Cultures with Gelatin Methacryloyl, Bovine Lactoferrin, and Bioactive Mesoporous Glass Scaffolds Loaded with Distinct Parsley Extracts.

The increasing interest in innovative solutions for addressing bone defects has driven research into the use of Bioactive Mesoporous Glasses (MBGs). These materials, distinguished by their well-ordered mesoporous structure, possess the capability to accommodate plant extracts with well-established osteogenic properties, including bovine lactoferrin (bLF), as part of their 3D scaffold composition. This harmonizes seamlessly with the ongoing advancements in the field of biomedicine. In this study, we fabricated 3D scaffolds utilizing MBGs loaded with extracts from parsley leaves (PL) and embryogenic cultures (EC), rich in bioactive compounds such as apigenin and kaempferol, which hold potential benefits for bone metabolism. Gelatin Methacryloyl (GelMa) served as the polymer, and bLF was included in the formulation. Cytocompatibility, Runx2 gene expression, ALP enzyme activity, and biomineralization were assessed in preosteoblastic MC3T3-E1 cell cultures. MBGs effectively integrated PL and EC extracts with loadings between 22.6 ± 0.1 and 43.6 ± 0.3 µM for PL and 26.3 ± 0.3 and 46.8 ± 0.4 µM for EC, ensuring cell viability through a release percentage between 28.3% and 59.9%. The incorporation of bLF in the 3D scaffold formulation showed significant differences compared to the control in all assays, even at concentrations below 0.2 µM. Combinations, especially PL + bLF at 0.19 µM, demonstrated additive potential, with superior biomineralization compared to EC. In summary, this study highlights the effectiveness of MBGs in incorporating PL and EC extracts, along with bLF, into 3D scaffolds. The results underscore cytocompatibility, osteogenic activity, and biomineralization, offering exciting potential for future in vivo applications.

Proof of Concept for Cell Culture-Based Coffee.

The global coffee production is facing serious challenges including land use, climate change, and sustainability while demand is rising. Cellular agriculture is a promising alternative to produce plant-based commodities such as coffee, which are conventionally produced by farming. In this study, the complex process of drying and roasting was adapted for bioreactor-grown coffee cells to generate a coffee-like aroma and flavor. The brews resulting from different roasting regimes were characterized with chemical and sensory evaluation-based approaches and compared to conventional coffee. Roasting clearly influenced the aroma profile. In contrast to conventional coffee, the dominant odor and flavor attributes were burned sugar-like and smoky but less roasted. The intensities of bitterness and sourness were similar to those of conventional coffee. The present results demonstrate a proof of concept for a cellular agriculture approach as an alternative coffee production platform and guide future optimization work.

Biochemical changes and enhanced accumulation of phenolic compounds in cell culture of Perilla frutescens (L.) by nano-chemical elicitation.

Perilla frutescens (L.) Britt is a renowned medicinal plant with pharmaceutically valuable phenolic acids and flavonoids. The present study was aimed to study the eliciting effect of silver and copper nanoparticles (AgNPs and CuNPs, 50 and 100 mg/L), and methyl jasmonate (MeJa, 50 and 100 µM) on the biochemical traits, the accumulation of phenolic compounds and antioxidative capacity of P. frutescens cell suspension culture. Suspension cells were obtained from friable calli derived from nodal explants in Murashige and Skoog (MS) liquid medium containing 1 mg/L 2,4-D and 1 mg/L BAP. The 21 days old cell suspension culture established from nodal explant derived callus supplemented with 100 mg/L MeJa resulted in the highest activity of catalase and guaiacol peroxidase enzymes, and CuNPs 100 mg/L treated cells indicated the maximum content of total phenol, total anthocyanin, superoxide dismutase, malondialdehyde, and H2O2. Also, the highest content of ferulic acid (1.41 ± 0.03, mg/g DW), rosmarinic acid (19.29 ± 0.12, mg/g DW), and phenylalanine ammonia-lyase (16.81 ± 0.18, U/mg protein) were observed with 100 mg/L CuNPs, exhibiting a total increase of 1.58-fold, 2.12-fold, and 1.51-fold, respectively, higher than untreated cells. On the other hand, AgNPs 100 mg/L treated cells indicated the most amounts of caffeic acid (0.57 ± 0.03, mg/g DW) and rutin (1.13 ± 0.07, mg/g DW), as well as the highest scavenging potential of free radicals. Overall, the results of the present study can be applied for the large-scale production of valuable phenolic acids and flavonoids from P. frutescens through CuNPs and AgNPs 100 mg/L elicited cell suspension cultures.

Effects of ceramide C2 application on human laryngeal carcinoma cells: a cell culture study.

OBJECTIVE: In the present study, we investigated the effects of Ceramide C2 application on human laryngeal carcinoma cells. MATERIALS AND METHODS: Human larynx epidermoid carcinoma HEp-2 (ATCC® CCL-23™) cells were purchased from the American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. Ceramide C2 was prepared, and further dilutions ranging from 3.13 to 100 µM were prepared in a fresh culture medium. Cells on 96 well plates were exposed to the prepared concentrations of ceramide C2 for 24 and 48 hours. Cytotoxicity evaluation was performed by MTT. Apoptosis profiles of HEp-2 cells were detected by annexin-V analysis. The activated caspases 3/7 on HEp-2 cells after ceramide C2 exposure were evaluated with flow cytometric analysis. The morphological changes on HEp-2 cells caused by ceramide C2 were evaluated by staining with phalloidine and acridine orange via confocal microscopy. For the Wound Healing Assay, HEp-2 cells were cultured in 6 well-plates until they became confluent. RESULTS: MTT cytotoxicity test findings revealed that the viability of human laryngeal carcinoma cells decreased with the increased application of ceramide C2 for 24 hours compared to untreated (control) cells. The highest growth inhibition by ceramide C2 for short-term application for 24 hours was detected at the highest concentration of ceramide C2 (100 µM). Annexin-V findings showed that 98.97 of HEp-2 cells were alive, and 1.63% were detected as early apoptosis for the control group. The results showed that ceramide C2 triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells of 61,40 compared to untreated HEp-2 cells. Cysteine proteases (caspases) 3/7 activation percentages of HEp-2 cells exposed to ceramide C2 for 24 hours were compared to control cells, and the morphology of HEp-2 cells was changed with clear apoptotic signs that underlined the cytotoxicity and pro-apoptotic activity of ceramide C2. Scratch Assay assessed the migration capability of HEp-2 cells before and after the exposure to ceramide C2. It showed that ceramide C2 reduced human laryngeal carcinoma cells' migration capability and proliferation for 24 hours. CONCLUSIONS: Based on all study findings, it can be considered that short-chain ceramide C2 exerted cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner and reduced the viability via inducing caspase-dependent apoptosis. The overall effect might be derived from the elevated intracellular ceramide levels by the exogenous application of ceramide C2. Consequently, it was concluded that ceramide C2 has good potential to cause cytotoxicity and apoptosis in human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anti-cancer drugs with high efficiency.

In vitro establishment of cell suspension culture of Ceropegia bulbosa for improved production of cerpegin content through elicitation of engineered carbon and ZnO nanoparticles.

 The present investigations aimed to conserve C. bulbosa a threatened plant species and for production of cerpegin through cell culture technology using ENP elicitation. Leaf explants were aseptically cultured with normal MS medium-supplemented PGRs BA and NAA various concentrations, and the best callus induction response was recorded on 4.5 + 4.5 µM. The prospective special effects of the ENPs on plant cell cultures are the key part of our study and used to evaluate leaf callus culture proliferation with the reduction of browning, establishment, biomass, and metabolite formation. The CNP concentrations (0, 2, 4, 8, 12 mg/l) are used for the callus proliferation and browning reduction. The cell suspension cultures are also established, and they were elicited with EZnONPs (0, 25, 50, 100, 150 mg/l) for evaluation of biomass, antioxidant, non-antioxidant enzyme activation, toxicity, ROS defense activation, and metabolite development in cell cultures. The metabolite extraction, UV, and NMR characterization confirm that the toxic and nontoxic effect of ENPs on leaf cell cultures varies with high to low concentration.

Quantitative assessment of cardiomyocyte mechanobiology through high-throughput cantilever-based functional well plate systems.

Proper regulation of the in vitro cell culture environment is essential for disease modelling and drug toxicity screening. The main limitation of well plates used for cell culture is that they cannot accurately maintain energy sources and compounds needed during cell growth. Herein, to understand the importance of perfusion in cardiomyocyte culture, changes in contractile force and heart rate during cardiomyocyte growth are systematically investigated, and the results are compared with those of a perfusion-free system. The proposed perfusion system consists of a Peltier refrigerator, a peristaltic pump, and a functional well plate. A functional well plate with 12 wells is made through injection moulding, with two tubes integrated in the cover for each well to continuously circulate the culture medium. The contractile force of cardiomyocytes growing on the cantilever surface is analysed through changes in cantilever displacement. The maturation of cardiomyocytes is evaluated through fluorescence staining and western blot; cardiomyocytes cultured in the perfusion system show greater maturity than those cultured in a manually replaced culture medium. The pH of the culture medium manually replaced at intervals of 3 days decreases to 6.8, resulting in an abnormal heartbeat, while cardiomyocytes cultured in the perfusion system maintained at pH 7.4 show improved contractility and a uniform heart rate. Two well-known ion channel blockers, verapamil and quinidine, are used to measure changes in the contractile force of cardiomyocytes from the two systems. Cardiomyocytes in the perfusion system show greater stability during drug toxicity screening, proving that the perfusion system provides a better environment for cell growth.