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International audience; Pancreatic cancer (PC) is a deadly cancer for which no diagnostic or prevention plan currently exists since this cancer is asymptomatic in its early development. The very specific and complex tumor microenvironment of PC is responsible for the unsuccessful delivery of therapeutics molecules to tumor cells. For high throughput testing of new molecules, there is a crucial need for more accurate preclinical model. For the first time, we integrated in a microfluidic system 5 major features of PC microenvironment: i) type-I collagen (CI), ii) hyaluronic acid (HA), iii) pancreatic stellate cells (SC), iv) interstitial fluid flow and v) nutrient and oxygen gradients. We showed long 3D culture (30 days). Furthermore, we implemented, thanks to the perfusion capability, the actual FOLFIRINOX protocol to assess the IC50 and we showed that dynamic conditions induce increased chemoresistance compared to 3D static.

The SH-SY5Y cell line is commonly used for the assessment of neurotoxicity in drug discovery. These neuroblastoma-derived cells can be differentiated into neurons using many methods. The present study has compared 24 of these differentiation methods on SH-SY5Y cells. After morphologic selection of the three most differentiating media (retinoic acid in 10% fetal bovine serum (FBS), staurosporine in 1% FBS medium, and cyclic adenosine monophosphate (cAMP) in B21-supplemented neurobasal medium), cells were analyzed for pan-neuronal and specific neuronal protein expression by fluorescent automated imaging. The response of SH-SY5Y to a set of compounds of known toxicity was examined in these culture conditions performed in 2D, and also in a 3D hyaluronic acid-based hydroscaffold™ which mimics the extracellular matrix. The extent of neuronal markers expression and the sensitivity to neurotoxic compounds varied according to the differentiation medium. The cAMP B21-supplemented neurobasal medium led to the higher neuronal differentiation, and the higher sensitivity to neurotoxic compounds. The culture in 3D modified the neurotoxic response, through a lower sensitivity of cells compared to the 2D culture. The in vitro differentiation environment influences the neurotoxic response of SH-SY5Y cells and thus should be considered carefully in research as well as in drug discovery.

Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1>GLUT3>GLUT5>GST1A>OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.

The most commonly used method to assess intestinal permeability is the measurement of compound flux across a Caco-2 cells monolayer by using Hanks balanced salt solution (HBSS)-like buffers. Nevertheless, lipophilic acid drugs are poorly or not at all soluble in these types of buffers and their adsorption on the transwell plate is commonly observed. To reduce adsorption and increase solubility, permeability assays need to be developed in conditions other than classic conditions for lipophilic compounds. The best model to increase recovery of lipophilic compounds was determined as fasted state simulated intestinal fluid (FaSSIF) in the apical compartment and HBSS with 1% bovine serum albumin (BSA) in basolateral compartment. This model allows a correlation between absorption on Caco-2 cells and absorbed fraction in humans. For 35 compounds, only 2 outliers were observed in the Caco-2 assay using the FaSSIF model. These two outliers were the same outlier compounds as those observed with a classic Caco-2 method. Furthermore, a permeability assay of Pgp substrates evidenced efflux transport in both models and addition of a Pgp inhibitor suppressed Pgp efflux transport. FaSSIF in the apical compartment and HBSS with 1% BSA in the basolateral compartment is the model of choice to predict in vivo absorption for lipophilic acid drugs.