A Shared Perspective on in Vitro and in Vivo Models to Assay Intestinal Transepithelial Transport of Food Compounds.

Assessing nutrient bioavailability is complex, as the process involves multiple digestion steps, several cellular environments, and regulatory-metabolic mechanisms. Several in vitro models of different physiological relevance are used to study nutrient absorption, providing significant challenges in data evaluation. However, such in vitro models are needed for mechanistic studies as well as to screen for biological functionality of the food structures designed. This collaborative work aims to put into perspective the wide-range of models to assay the permeability of food compounds considering the particular nature of the different molecules, and, where possible, in vivo data are provided for comparison.

Coupling in vitro food digestion with in vitro epithelial absorption; recommendations for biocompatibility.

As food transits the gastrointestinal tract, food structures are disrupted and nutrients are absorbed across the gut barrier. In the past decade, great efforts have focused on the creation of a consensus gastrointestinal digestion protocol (i.e., INFOGEST method) to mimic digestion in the upper gut. However, to better determine the fate of food components, it is also critical to mimic food absorption in vitro. This is usually performed by treating polarized epithelial cells (i.e., differentiated Caco-2 monolayers) with food digesta. This food digesta contains digestive enzymes and bile salts, and if following the INFOGEST protocol, at concentrations that although physiologically relevant are harmful to cells. The lack of a harmonized protocol on how to prepare the food digesta samples for downstream Caco-2 studies creates challenges in comparing inter laboratory results. This article aims to critically review the current detoxification practices, highlight potential routes and their limitations, and recommend common approaches to ensure food digesta is biocompatible with Caco-2 monolayers. Our ultimate aim is to agree a harmonized consensus protocol or framework for in vitro studies focused on the absorption of food components across the intestinal barrier.

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.

Sodium butyrate converts Caco-2 monolayers into a leaky but healthy intestinal barrier resembling that of a newborn infant.

A simple and reliable in vitro model of the infant intestinal barrier is needed to study nutrient absorption and drug permeability specifically for this life stage. This study investigated the treatment of 20 day old differentiated Caco-2 monolayers with sodium butyrate at various concentrations (0-250 mM). Monolayer integrity, cytotoxicity, permeability and inflammatory response were tracked. An intestinal barrier model, with infant gut characteristics, was developed based on the treatment of mature monolayers with 125 mM sodium butyrate for 24 h. Such treatment was not cytotoxic but caused a stable transepithelial electrical resistance value of 408 ± 52 Ω cm2. The ratio of lactulose to mannitol transport across the intestinal barrier increased 1.79-fold. Redistribution of the tight junction proteins, occludin and ZO-1, in response to sodium butyrate treatment was visualized with immunofluorescence. Levels of the cytokines, TNF-α and IL-6, although modestly increased did not indicate an inflammatory response by Caco-2 to sodium butyrate. This intestinal barrier demonstrated physiologically relevant transport rates for dairy protein of 0.01-0.06%, suggesting it may be used to track permeability of proteins in infant nutritional products.

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.

Irish Cheddar cheese increases glucagon-like peptide-1 secretion in vitro but bioactivity is lost during gut transit.

Appetite can be effectively reduced by targeting the production, secretion, circulation time or receptor of the enteric satiety hormone glucagon-like peptide-1 (GLP-1). The objective of this study was to investigate the potency of Irish Cheddar cheeses to modulate GLP-1 levels. Nine out of ten water-soluble extracts (WSEs) of representative Irish Cheddar cheeses, post 6 months ripening, significantly (p < 0.05) stimulated active GLP-1 secretion from the mouse enteroendocrine cell line STC-1. This secretion was associated with protein content and ripening time of cheese. C-57BL/6 mice (n = 9/10), who received the most potent sample, C2-WSE-8 M, had a significantly reduced cumulative food intake at 6 h compared to control (p < 0.05), but not overall treatment × time effect over a 7 h period. Simulated in vitro gastrointestinal digestion, that models the upper human gut, indicated loss of GLP-1 stimulating activity once C2-WSE-8M entered the intestinal phase, suggesting efficacy of C2-WSE-8M will depend on protection during gut transit.

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

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. 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. 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.

Comparison of the three optical platforms for measurement of cellular respiration.

We compared three optical platforms for measurement of cellular respiration: absolute oxygen consumption rates (OCRs) in hermetically sealed microcuvettes, relative OCRs measured in a 96-well plate with oil seal, and steady-state oxygenation of cells in an open 96-well plate. Using mouse embryonic fibroblasts cell line, the phosphorescent intracellular O2 probe MitoXpress-Intra, and time-resolved fluorescence reader, we determined algorithms for conversion of relative OCRs and cell oxygenation into absolute OCRs, thereby allowing simple high-throughput measurement of absolute OCR values.

Imaging oxygen in neural cell and tissue models by means of anionic cell-permeable phosphorescent nanoparticles.

Cell-permeable phosphorescent probes enable the study of cell and tissue oxygenation, bioenergetics, metabolism, and pathological states such as stroke and hypoxia. A number of such probes have been described in recent years, the majority consisting of cationic small molecule and nanoparticle structures. While these probes continue to advance, adequate staining for the study of certain cell types using live imaging techniques remains elusive; this is particularly true for neural cells. Here we introduce novel probes for the analysis of neural cells and tissues: negatively charged poly(methyl methacrylate-co-methacrylic acid)-based nanoparticles impregnated with a phosphorescent Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye (this form is referred to as PA1), and with an additional reference/antennae dye poly(9,9-diheptylfluorene-alt-9,9-di-p-tolyl-9H-fluorene) (this form is referred to as PA2). PA1 and PA2 are internalised by endocytosis, result in efficient staining in primary neurons, astrocytes, and PC12 cells and multi-cellular aggregates, and allow for the monitoring of local O(2) levels on a time-resolved fluorescence plate reader and PLIM microscope. PA2 also efficiently stains rat brain slices and permits detailed O(2) imaging experiments using both one and two-photon intensity-based modes and PLIM modes. Multiplexed analysis of embryonic rat brain slices reveals age-dependent staining patterns for PA2 and a highly heterogeneous distribution of O(2) in tissues, which we relate to the localisation of specific progenitor cell populations. Overall, these anionic probes are useful for sensing O(2) levels in various cells and tissues, particularly in neural cells, and facilitate high-resolution imaging of O(2) in 3D tissue models.

Small molecule phosphorescent probes for O2 imaging in 3D tissue models.

Monitoring of oxygenation is important for physiological experiments investigating the growth, differentiation and function of individual cells in 3D tissue models. Phosphorescence based O2 sensing and imaging potentially allow this task; however, current probes do not provide the desired bio-distribution and analytical performance. We present several new cell-penetrating phosphorescent conjugates of a Pt(ii)-tetrakis(pentafluorophenyl)porphine (PtPFPP) dye produced by click-modification with thiols, and perform their evaluation as O2 imaging probes for 3D tissue models. The hydrophilic glucose (Pt-Glc) and galactose (Pt-Gal) conjugates demonstrated minimal aggregation and self-quenching in aqueous media, and efficient in-depth staining of different cell types and multi-cellular aggregates at working concentrations ≤10 µM. The Pt-Glc probe was applied in high-resolution phosphorescence lifetime based O2 imaging (PLIM) in multi-cellular spheroids of cancer cells (PC12), primary neural cells (neurospheres) and slices of brain tissue, where it showed good analytical performance, minimal effects on cell viability and appropriate responses to O2 with phosphorescence lifetimes changing from 20 µs in air-saturated to 57 µs under deoxygenated conditions. In contrast, mono- and tetra-substituted oligoarginine conjugates of PtPFPP showed marked aggregation and unstable photophysical properties precluding their use as O2 sensing probes.

Measurement of cell respiration and oxygenation in standard multichannel biochips using phosphorescent O2-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 (µ-slides, Ibidi) and phosphorescent O2 sensitive probes. This platform was evaluated with both extracellular and intracellular O2 probes, several different cell types and treatments including mitochondrial uncoupling and inhibition, depletion of extracellular Ca(2+) and inhibition of V-ATPase and histone deacetylases. The results show that compared to the standard microwell plates currently used, the µ-slide platform provides facile O2 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.