Culture of primary human meibomian gland cells from surgically excised eyelid tissue.

Meibomian gland dysfunction is one of the most common ocular diseases, with therapeutic treatment being primarily palliative due to our incomplete understanding of meibomian gland (MG) pathophysiology. To progress in vitro studies of human MG, this study describes a comprehensive protocol, with detailed troubleshooting, for the successful isolation, cultivation and cryopreservation of primary MG cells using biopsy-size segments of human eyelid tissue that would otherwise be discarded during surgery. MG acini were isolated and used to establish and propagate lipid-producing primary human MG cells. The primary cell viability during culture procedure was maintained through the application of Rho-associated coiled-coil containing protein kinase inhibitor (Y-27632, 10 µM) and collagen I from rat tails. Transcriptomic analysis of differentiated primary human MG cells confirmed cell origin and revealed high-level expression of many lipogenesis-related genes such as stearoyl-CoA desaturase (SCD), ELOVL Fatty Acid Elongase 1 (ELOVL1) and fatty acid synthase (FASN). Primary tarsal plate fibroblasts were also successfully isolated, cultured and cryopreserved. Established primary human MG cells and tarsal plate fibroblasts presented in this study have potential for applications in 3D models and bioengineered tissue that facilitate research in understanding of MG biology and pathophysiology.

Analysis of sex steroids in human tears using LC-MS and GC-MS: Considerations and developments to improve method sensitivity and accuracy.

Sex steroids play a role in regulation of tear film function and may exert their action locally at the ocular surface. However, measurement of sex steroids in tears is difficult due to small-volume tear samples and very low concentrations of the hormones. This short communication highlights what has been achieved to date in the analysis of tear sex steroids using ultra-performance LC-MS (UPLC-MS) as previously published, and reports further and more recent investigations toward optimising mass spectrometry method sensitivity and accuracy. The published UPLC-MS method successfully measured progesterone, androsterone glucuronide and 5α-androstane-3α,17ß-diol in pooled basal tears of postmenopausal women, and fourteen sex steroid standards in methanol. Limitations included sub-optimal limits of detection (LOD) and lower limits of quantification (LLOQ) for some analytes (particularly oestrogens), exclusion of sample matrix effects and no use of internal standards. This update reports on further experiments carried out to improve sensitivity and accuracy. Sample matrix effects, internal standard spiking, and derivatisation with dansyl chloride and oximes were investigated. Dansylation significantly improved the LOD and LLOQ of oestrogens and their metabolites, by a factor of 10 for oestradiol and a factor of 5 for oestrone, but sensitivity of this updated method is not sufficient however for analysis of these oestrogens in human tears. Using gas chromatography-mass spectrometry (GC-MS) as an alternative technique to LC-MS, improved sensitivity for derivatised oestradiol is reported. This work demonstrates the need to develop higher sensitivity methods and points researchers towards specific MS ionisation techniques for future analysis of sex steroids in tears, in order to progress current understanding of the role of sex steroids in tear function and dry eye.

Human meibomian gland epithelial cell culture models: Current progress, challenges, and future directions.

The widely used immortalised human meibomian gland epithelia cell (iHMGEC) line has made possible extensive studies of the biology and pathophysiology of meibomian glands (MG). Tissue culture protocols for iHMGEC have been revised and modified to optimise the growth conditions for cell differentiation and lipid accumulation. iHMGEC proliferate in serum-free medium but require serum or other appropriate exogenous factors to differentiate. Several supplements can enhance differentiation and neutral lipid accumulation in iHMGEC grown in serum-containing medium. In serum-free medium, rosiglitazone, a peroxisome proliferator activator receptor-γ (PPARγ) agonist, is reported to induce iHMGEC differentiation, neutral lipid accumulation and expression of key biomarkers of differentiation. iHMGEC cultured in serum-containing medium under hypoxia or with azithromycin increases DNAse 2 activity, a biomarker of terminal differentiation in sebocytes. The production of lipids with composition similar to meibum has not been observed in vitro and this remains a major challenge for iHMGEC culture. Innovative methodologies such as 3D ex vivo culture of MG and generation of MG organoids from stem cells are important for further developing a model that more closely mimics the in vivo biology of human MG and to facilitate the next generation of studies of MG disease and dry eye.

Semi-quantification of lipids in human meibomian gland epithelial cells using dual staining microplate assays.

Two spectrophotometric microplate assays with dual staining for either fluorescent Nile red (NR) plus 4,6-diamidino-2-phenylindole (DAPI) or non-fluorescent Oil red O (ORO) plus Crystal violet (CV) were applied and optimised to evaluate the lipid producing capacity of immortalised human meibomian gland epithelial cells (iHMGEC). Cells were treated with rosiglitazone (Rosi, 10-50 µM), a known lipid producing inducer for iHMGEC, and were analysed for lipids using the NR-DAPI and ORO-CV microplate assays. The lipid producing capacity of iHMGEC after each treatment was determined by normalising lipid quantity (measured with NR or ORO) to cell number (measured with DAPI or CV). The dye concentrations of NR 1 µg/mL, DAPI 5 µg/mL, ORO 0.3% (v/v) and CV 0.5% (v/v), provided optimal linearity and coverage of signals over a range of cell densities (corresponding to 10-100% cell confluence). Both NR-DAPI and ORO-CV showed a dose-dependent effect of Rosi on lipid production in iHMGEC, consistent with the results reported previously using traditional microscopic imaging methods. The microplate assays offer a rapid, high throughput and objective measurement of the amount of lipids in iHMGEC (and potentially other lipid-producing cells) and can be used for screening the effects of biological agents or incubation changes on lipid production in cells in future studies.

Anthocyanin-based sensors derived from food waste as an active use-by date indicator for milk.

Active use-by date (AUBD) or freshness indicators hold great potential to reduce food waste. Herein, we develop an anthocyanin AUBD indicator that is capable of discriminating between fresh, spoiling, and spoiled milk. The sensor undergoes a visible blue to purple to pink color change in response to lactic acid, which is an indicator of microbial spoilage in milk. Anthocyanin is cast into a range of materials and the composite's suitability to monitor pH changes (pH 6.8 fresh milk vs pH 4.0 spoiled milk) is assessed. Of the materials studied, an anthocyanin-agarose film is nominated as the optimum materials with the best colorimetric performance. We introduce a new method to quantify anthocyanin color change by measuring red chromatic shift by digital analysis. The anthocyanin sensors will provide a real-time indication of actual milk quality, surpassing the function of traditional date marking tools that provide an indication of the expected shelf life.

Co-ingestion of red cabbage with cherry tomato enhances digestive bioaccessibility of anthocyanins but decreases carotenoid bioaccessibility after simulated in vitro gastro-intestinal digestion.

The effects of co-digestion of red cabbage with carrot, baby spinach and/or cherry tomato on the bioaccessibility of anthocyanins and carotenoids such as α-carotene, ß-carotene, lutein and lycopene were examined using a simulated in vitro gastro-intestinal digestion model. The individual vegetables and their mixtures were digested with and without added a standardised salad dressing. Bioaccessibility of total anthocyanins was enhanced by 10-15% (p < 0.05) when red cabbage was co-digested with the carotenoid-rich vegetables, except with carrot. In contrast, the co-digestion of red cabbage with carrot decreased bioaccessibility of total carotenoids by 21-33% (p < 0.05), and with cherry tomato by 42-56% (p < 0.05). The bioaccessibility of a given carotenoid varied depending on the vegetable matrix. Among the tested vegetable mixtures, red cabbage and baby spinach when co-digested demonstrated that anthocyanins and carotenoids were equally bioaccessible (total anthocyanin bioaccessibility of 62-66% and total carotenoid bioaccessibility of 66%).

Effects on intestinal cellular bioaccessibility of carotenoids and cellular biological activity as a consequence of co-ingestion of anthocyanin- and carotenoid-rich vegetables.

The effects of co-digestion of a carotenoid-rich vegetable such as carrot, cherry tomato or baby spinach with an anthocyanin-rich vegetable such as red cabbage with and without salad dressing on the intestinal cellular bioaccessibility (cBAC) of carotenoids and the resultant cellular antioxidant and anti-inflammatory activities were investigated. The % cBAC of lutein from the tested vegetables was 0.23-1.42%, lycopene 0.07-0.39%, α-carotene 0.01-0.12% and ß-carotene 0.03-0.61% respectively. The % cBAC of each of these carotenoids from the co-digested vegetables was significantly higher (p < 0.05) than from carrot, cherry tomato or baby spinach digested alone. % cBAC of total carotenoids was significantly increased by 46-191% (p < 0.05) as a result of the co-digestion. The vegetable co-digestion did not result in any impairment on the resultant cellular anti-oxidation and anti-inflammation (NO, IL-8 secretion). Among the tested vegetables, baby spinach co-digested with red cabbage showed synergistic bioactivities in all tested assays.

Interferences of anthocyanins with the uptake of lycopene in Caco-2 cells, and their interactive effects on anti-oxidation and anti-inflammation in vitro and ex vivo.

Lycopene was combined with the glucosides of each of the six common anthocyanidins at 3 different ratios to investigate their interactions on antioxidant and anti-inflammatory activities, and cellular uptake. The bioactivity interaction between lycopene and anthocyanins was studied in both chemical and cellular models. Anti-oxidative synergy was not seen in any of the tested lycopene-anthocyanin mixtures, nor in the models studied. When lycopene was paired with the methoxylated anthocyanins, the anti-inflammatory effect on the inhibition of the cytokine IL-8, which is a pro-inflammatory biomarker, was increased by 15-69% of the expected additive activity, indicating synergistic interaction between the compounds. The cellular uptake of lycopene was significantly impaired by the presence of the anthocyanins: reduced by 50-80% at the lycopene: anthocyanin combinatory ratios of 2.5:7.5 µM (1:3) or 5:5 µM (1:1). The reduced intracellular lycopene content might be partly responsible for the antagonistic cellular antioxidant property seen in some of the tested mixtures.

Effect of Different Anthocyanidin Glucosides on Lutein Uptake by Caco-2 Cells, and Their Combined Activities on Anti-Oxidation and Anti-Inflammation In Vitro and Ex Vivo.

The interactive effects on anti-oxidation and anti-inflammation of lutein combined with each of the six common anthocyanidin glucosides were studied in both chemical and cellular systems. The combined phytochemicals showed an antagonism in the inhibition of lipid oxidation in a liposomal membrane, but showed an additive effect on cellular antioxidant activity in Caco-2 cells. Lutein was an active lipoxygenase inhibitor at 2⁻12 µM while anthocyanins were inactive. The concentration of lutein when it was used in combination with anthocyanins was 25⁻54% higher than when lutein was used alone (i.e., IC50 = 1.2 µM) to induce 50% of lipoxygenase inhibition. Only the combination of lutein with malvidin-3-glucoside showed anti-inflammatory synergy in the suppression of interleukin-8, and the synergy was seen at all three ratios tested. Some mixtures, however, showed anti-inflammatory antagonism. The presence of anthocyanins (5⁻7.5 µM) did not affect lutein uptake (2.5⁻5 µM) by Caco-2 cells.

Interactions between phytochemicals from fruits and vegetables: Effects on bioactivities and bioavailability.

The combinations of two or more phytochemicals bring about changes in the ultimate biological effects and/or the bioavailability of each component. A number of mixtures of pure bioactive compounds or phytochemical-containing plant extracts provide synergy with regard to antioxidant status, anti-inflammation, anti-cancer and chemoprevention of several oxidative stress and metabolic disorders in vitro. The biological activities of food phytochemicals depend upon their bioaccessibility and bioavailability which can be affected by the presence of other food components including other bioactive constituents. The interactions between phytochemicals during intestinal absorption could result in changes in the bioavailability of the compounds, which in turn affects the intensity of their bioactivities. This paper provides an overview of combined biological effects of phytochemical mixtures derived from fruits and vegetables with a focus on anti-oxidative, anti-inflammatory and anti-carcinogenic activities. The bioavailability impairment or enhancement caused by the co-consumption of dietary phytochemicals is also discussed. Finally, research gaps for future studies on phytochemical interactions are identified.