In vitro models that better reflect in vivo epithelial barrier (patho-)physiology are urgently required to predict adverse drug effects. Here we introduce extracellular matrix-supported intestinal tubules in perfused microfluidic devices, exhibiting tissue polarization and transporter expression. Forty leak-tight tubules are cultured in parallel on a single plate and their response to pharmacological stimuli is recorded over 125 h using automated imaging techniques. A study comprising 357 gut tubes is performed, of which 93% are leak tight before exposure. EC50-time curves could be extracted that provide insight into both concentration and exposure time response. Full compatibility with standard equipment and user-friendly operation make this Organ-on-a-Chip platform readily applicable in routine laboratories.Efforts to determine the effects of drugs on epithelial barriers could benefit from better in vitro models. Here the authors develop a microfluidic device supporting the growth and function of extracellular matrix-supported intestinal tubules, and evaluate the effect of staurosporine and acetylsalicylic acid on barrier integrity.
Traditionally, to quantify permeability of a biological barrier, the initial slope is used, based on the assumption of sink condition (concentration of the donor is constant, and the receiver increases less than 10%). With on-a-chip barrier models, this assumption fails in cell-free or leaky conditions, which requires the use of the exact solution. To encounter a time delay from performing the assay and acquiring the data, we present a protocol with the exact equation modified to incorporate a time offset.
The microstructure and permeability of rehydrated 20−100 μm thick partially coalesced (vinyl-actetate acrylic copolymer) SF091 latex coatings and a 118 μm thick model trilayer biocatalytic coating consisting of two sealant SF091 layers containing a middle layer of viable E. coli HB101 + latex were studied as delaminated films in a diffusion apparatus with KNO3 as the diffussant. The permeability of the hydrated coatings is due to diffusive transport through the pore space between the partially coalesced SF091 latex particles. Coating microstructure was visualized by fast freeze cryogenic scanning electron microscopy (cryo-SEM). The effective diffusion coefficient of SF091 latex coatings (diffusive permeability/film thickness) was determined as the ratio of the effective diffusivity of KNO3 to its diffusivity in water (Deff/D). Polymer particle coalescence was arrested by two methods to increase coating permeability. The first used glycerol with coating drying at 4 °C, near the glass transition temperature (Tg). The second method used sucrose or trehalose as a filler to arrest coalescence; the filler was then dissolved away. Deff/D was measured as a function of film thickness; content of glycerol, sucrose, and trehalose; drying time; and rehydration time. Deff/D varied from 3 × 10−4 for unmodified SF091 coatings to 6.8 × 10−2 for coatings containing sucrose. Deff/D was reduced by the flattening of latex particles against the surface of the solid substrate, as well as by the presence of the colloid stabilizer hydroxyethylcellulose (HEC). When corrected for the flattened particle layer, Deff/D of HEC-free coatings was as high as 0.20, which agreed with the value prediced from analysis of cryo-SEM images of the coat surface. Deff/D decreased by one-half in approximately 5 days in rehydrated SF091 coatings, indicating that significant wet coalescence occurs after glycerol, sucrose, or trehalose are leached from the films. Deff/D of SF091 latex trilayer coatings containing viable E. coliHB101 cells decreased as cell loading was increased from 2.2 × 10−2 for 64 g dry cell weight per liter of coat volume to 5 × 10−3 for 151 g DCW/L of coat volume. The reduction in coating permeability with increasing cell loading is predicted by Maxwell's equation for Deff/D in periodic composites.
PRL1 and PRL3, members of the protein tyrosine phosphatase family, have been associated with cancer metastasis and poor prognosis.Despite extensive research on their protein phosphatase activity, their potential role as lipid phosphatases remains elusive.Methods: We conducted comprehensive investigations to elucidate the lipid phosphatase activity of PRL1 and PRL3 using a combination of cellular assays, biochemical analyses, and protein interactome profiling.Functional studies were performed to delineate the impact of PRL1/3 on macropinocytosis and its implications in cancer biology.Results: Our study has identified PRL1 and PRL3 as lipid phosphatases that interact with phosphoinositide (PIP) lipids, converting PI(3,4)P2 and PI(3,5)P2 into PI(3)P on the cellular membranes.These enzymatic activities of PRLs promote the formation of membrane ruffles, membrane blebbing and subsequent macropinocytosis, facilitating nutrient extraction, cell migration, and invasion, thereby contributing to tumor development.These enzymatic activities of PRLs promote the formation of membrane ruffles, membrane blebbing and subsequent macropinocytosis.Additionally, we found a correlation between PRL1/3 expression and glioma development, suggesting their involvement in glioma progression.Conclusions: Combining with the knowledge that PRLs have been identified to be involved in mTOR, EGFR and autophagy, here we concluded the physiological role of PRL1/3 in orchestrating the nutrient sensing, absorbing and recycling via regulating macropinocytosis through its lipid phosphatase activity.This mechanism could be exploited by tumor cells facing a nutrient-depleted microenvironment, highlighting the potential therapeutic significance of targeting PRL1/3-mediated macropinocytosis in cancer treatment.
Breast cancer is the most common invasive cancer among women. Currently, there are only a few models used for therapy selection, and they are often poor predictors of therapeutic response or take months to set up and assay. In this report, we introduce a microfluidic OrganoPlate® platform for extracellular matrix (ECM) embedded tumor culture under perfusion as an initial study designed to investigate the feasibility of adapting this technology for therapy selection. The triple negative breast cancer cell lines MDA-MB-453, MDA-MB-231 and HCC1937 were selected based on their different BRCA1 and P53 status, and were seeded in the platform. We evaluate seeding densities, ECM composition (Matrigel®, BME2rgf, collagen I) and biomechanical (perfusion vs static) conditions. We then exposed the cells to a series of anti-cancer drugs (paclitaxel, olaparib, cisplatin) and compared their responses to those in 2D cultures. Finally, we generated cisplatin dose responses in 3D cultures of breast cancer cells derived from 2 PDX models. The microfluidic platform allows the simultaneous culture of 96 perfused micro tissues, using limited amounts of material, enabling drug screening of patient-derived material. 3D cell culture viability is improved by constant perfusion of the medium. Furthermore, the drug response of these triple negative breast cancer cells was attenuated by culture in 3D and differed from that observed in 2D substrates. We have investigated the use of a high-throughput organ-on-a-chip platform to select therapies. Our results have raised the possibility to use this technology in personalized medicine to support selection of appropriate drugs and to predict response to therapy in a real time fashion.
Micromolded hydrogels lose non-fouling properties enabling cell adhesion and migration on the gel (left). We develop a method to regenerate the cell resistance of the gels resulting in robust resistance to cell adhesion (right).
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