New phosphorescence based probes and techniques for the analysis of cellular oxygen and respiration

Real time monitoring of oxygenation and respiration is on the cutting edge of bioanalysis, including studies of cell metabolism, bioenergetics, mitochondrial function and drug toxicity. This thesis presents the development and evaluation of new luminescent probes and techniques for intracellular O2 sensing and imaging. A new oxygen consumption rate (OCR) platform based on the commercial microfluidic perfusion channel μ-slides compatible with extra- and intracellular O2 sensitive probes, different cell lines and measurement conditions was developed. The design of semi-closed channels allowed cell treatments, multiplexing with other assays and two-fold higher sensitivity to compare with microtiter plate. We compared three common OCR platforms: hermetically sealed quartz cuvettes for absolute OCRs, partially sealed with mineral oil 96-WPs for relative OCRs, and open 96-WPs for local cell oxygenation. Both 96-WP platforms were calibrated against absolute OCR platform with MEF cell line, phosphorescent O2 probe MitoXpress-Intra and time-resolved fluorescence reader. Found correlations allow tracing of cell respiration over time in a high throughput format with the possibility of cell stimulation and of changing measurement conditions. A new multimodal intracellular O2 probe, based on the phosphorescent reporter dye PtTFPP, fluorescent FRET donor and two-photon antennae PFO and cationic nanoparticles RL-100 was described. This probe, called MM2, possesses high brightness, photo- and chemical stability, low toxicity, efficient cell staining and high-resolution intracellular O2 imaging with 2D and 3D cell cultures in intensity, ratiometric and lifetime-based modalities with luminescence readers and FLIM microscopes. Extended range of O2 sensitive probes was designed and studied in order to optimize their spectral characteristics and intracellular targeting, using different NPs materials, delivery vectors, ratiometric pairs and IR dyes. The presented improvements provide useful tool for high sensitive monitoring and imaging of intracellular O2 in different measurement formats with wide range of physiological applications

Physiological Gut Oxygenation Alters GLP‐1 Secretion from the Enteroendocrine Cell Line STC‐1

peer-reviewed1 Scope Enteroendocrine cell lines are routinely assayed in simple buffers at ≈20% oxygen to screen foods for bioactives that boost satiety hormone levels. However, in vivo, enteroendocrine cells are exposed to different phases of food digestion and function at low oxygen concentration, ranging from 7.5% in the stomach to 0.5% in the colon–rectal junction. 2 Methods and results The objective of this study is to investigate the effect of physiologically relevant O2 concentrations of the gut on the production and secretion of the satiety hormone, glucagon‐like peptide 1 (GLP‐1), from the murine enteroendocrine cell line, secretin tumor cell line (STC‐1), in response to dairy macronutrients as they transit the gut. GLP‐1 exocytosis from STC‐1 cells is influenced by both oxygen concentration and by individual macronutrients. At low oxygen, STC‐1 cell viability is significantly improved for all macronutrient stimulations and cyclic adenosine monophosphate levels are dampened. GLP‐1 secretion from STC‐1 cells is influenced by both the phase of yogurt digestion and corresponding O2 concentration. Atmospheric oxygen at 4.5% combined with upper gastric digesta, which simulates ileum conditions, yields the highest GLP‐1 response. 3 Conclusion This demonstrates the importance of considering physiological oxygen levels and food digestion along gastrointestinal tract for reliable in vitro analysis of gut hormone secretion

Dairy-derived peptides for satiety

peer-reviewedSatiety hormones produced in the gastrointestinal tract are key players in influencing appetite and food intake. Dairy proteins that target these gastric signals have the potential to make one feel ‘fuller for longer’. While effects of whey and casein on appetite and food intake are well documented, this review focuses on individual dairy peptides. The evidence of these peptide bioactives on satiety signaling in vitro using cellular models and in vivo via intervention trials is summarized. Dairy protein hydrolysates are also reviewed for their satiating properties. How their efficacy compares to other notable food derived peptides and how this efficacy can be lost, bolstered or protected during gut transit is also summarized

Measurement of cell respiration and oxygenation in standard multichannel biochips using phosphorescent O-2-sensitive probes

Measurement of cell oxygenation and oxygen consumption is useful for studies of cell bioenergetics, metabolism, mitochondrial function, drug toxicity and common pathophysiological conditions. Here we present a new platform for such applications which uses commercial multichannel biochips (mu-slides, Ibidi) and phosphorescent O-2 sensitive probes. This platform was evaluated with both extracellular and intracellular O-2 probes, several different cell types and treatments including mitochondrial uncoupling and inhibition, depletion of extracellular Ca2+ and inhibition of V-ATPase and histone deacetylases. The results show that compared to the standard microwell plates currently used, the mu-slide platform provides facile O-2 measurements with both suspension and adherent cells, higher sensitivity and reproducibility, and faster measurement time. It also allows re-perfusion and multiple treatments of cells and multi-parametric analyses in conjunction with other probes. Optical measurements are conducted on standard fluorescence readers and microscopes

Affordable oxygen microscopy-assisted biofabrication of multicellular spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source

Effects of Protein-Derived Amino Acid Modification Products Present in Infant Formula on Metabolic Function, Oxidative Stress, and Intestinal Permeability in Cell Models

Proteins present in infant formulas are modified by oxidation and glycation during processing. Modified amino acid residues released from proteins may be absorbed in the gastrointestinal tract, and pose a health risk to infants. In this study, the markers of glycation furosine (1.7–3.5 μg per milligram of protein) and Nε-(carboxymethyl)­lysine (28–81 ng per milligram of protein) were quantitated in infant formulas. The effects of these species, and other amino acid modifications, at the levels detected in infant formulas, on 3T3-L1 (murine preadipocyte) and Caco-2 (human intestinal epithelial) cells were assessed. Incubation of 3T3-L1 cells for 48 h with amino acid side chain oxidation and glycation products (1 and 10 μM) resulted in a loss (up to 40%, p < 0.05) of cell thiols and decreased metabolic activity compared with those of the controls. In contrast, Caco-2 cells showed a stimulation (10–50%, p < 0.05) of cellular metabolism on exposure to these products for 24 or 48 h. A 28% (p < 0.05) increase in protein carbonyls was detected upon incubation with 200 μM modified amino acids for 48 h, although no alteration in transepithelial electrical resistance was detected. Oxidation products were detected in the basolateral compartments of Caco-2 monolayers when modified amino acids were applied to the apical side, consistent with limited permeability (up to 3.4%) across the monolayer. These data indicate that modified amino acids present in infant formulas can induce effects on different cell types, with evidence of bioavailability and induction of cellular stress. This may lead to potential health risks for infants consistently exposed to high levels of infant formulas