A New Microfluidic Device to Facilitate Functional Precision Medicine Assays.

Functional precision medicine (FPM) has emerged as a new approach to improve cancer treatment. Despite its potential, FPM assays present important limitations such as the number of cells and trained personnel required. To overcome these impediments, here we describe a novel microfluidic platform that can be used to perform FPM assays, optimizing the use of primary cancer cells and simplifying the process by using microfluidics to automatize the process.

Parallel detection of chemical reactions in a microfluidic platform using hyperpolarized nuclear magnetic resonance.

The sensitivity of NMR may be enhanced by more than four orders of magnitude via dissolution dynamic nuclear polarization (dDNP), potentially allowing real-time, in situ analysis of chemical reactions. However, there has been no widespread use of the technique for this application and the major limitation has been the low experimental throughput caused by the time-consuming polarization build-up process at cryogenic temperatures and fast decay of the hyper-intense signal post dissolution. To overcome this limitation, we have developed a microfluidic device compatible with dDNP-MR spectroscopic imaging methods for detection of reactants and products in chemical reactions in which up to 8 reactions can be measured simultaneously using a single dDNP sample. Multiple MR spectroscopic data sets can be generated under the same exact conditions of hyperpolarized solute polarization, concentration, pH, and temperature. A proof-of-concept for the technology is demonstrated by identifying the reactants in the decarboxylation of pyruvate via hydrogen peroxide (e.g. 2-hydroperoxy-2-hydroxypropanoate, peroxymonocarbonate and CO2). dDNP-MR allows tracing of fast chemical reactions that would be barely detectable at thermal equilibrium by MR. We envisage that dDNP-MR spectroscopic imaging combined with microfluidics will provide a new high-throughput method for dDNP enhanced MR analysis of multiple components in chemical reactions and for non-destructive in situ metabolic analysis of hyperpolarized substrates in biological samples for laboratory and preclinical research.

Microfluidic-based dynamic BH3 profiling predicts anticancer treatment efficacy.

Precision medicine is starting to incorporate functional assays to evaluate anticancer agents on patient-isolated tissues or cells to select for the most effective. Among these new technologies, dynamic BH3 profiling (DBP) has emerged and extensively been used to predict treatment efficacy in different types of cancer. DBP uses synthetic BH3 peptides to measure early apoptotic events ('priming') and anticipate therapy-induced cell death leading to tumor elimination. This predictive functional assay presents multiple advantages but a critical limitation: the cell number requirement, that limits drug screening on patient samples, especially in solid tumors. To solve this problem, we developed an innovative microfluidic-based DBP (µDBP) device that overcomes tissue limitations on primary samples. We used microfluidic chips to generate a gradient of BIM BH3 peptide, compared it with the standard flow cytometry based DBP, and tested different anticancer treatments. We first examined this new technology's predictive capacity using gastrointestinal stromal tumor (GIST) cell lines, by comparing imatinib sensitive and resistant cells, and we could detect differences in apoptotic priming and anticipate cytotoxicity. We then validated µDBP on a refractory GIST patient sample and identified that the combination of dactolisib and venetoclax increased apoptotic priming. In summary, this new technology could represent an important advance for precision medicine by providing a fast, easy-to-use and scalable microfluidic device to perform DBP in situ as a routine assay to identify the best treatment for cancer patients.

Tight junction channel regulation by interclaudin interference.

Tight junctions form selectively permeable seals across the paracellular space. Both barrier function and selective permeability have been attributed to members of the claudin protein family, which can be categorized as pore-forming or barrier-forming. Here, we show that claudin-4, a prototypic barrier-forming claudin, reduces paracellular permeability by a previously unrecognized mechanism. Claudin-4 knockout or overexpression has minimal effects on tight junction permeability in the absence of pore-forming claudins. However, claudin-4 selectively inhibits flux across cation channels formed by claudins 2 or 15. Claudin-4-induced loss of claudin channel function is accompanied by reduced anchoring and subsequent endocytosis of pore-forming claudins. Analyses in nonepithelial cells show that claudin-4, which is incapable of independent polymerization, disrupts polymeric strands and higher order meshworks formed by claudins 2, 7, 15, and 19. This process of interclaudin interference, in which one claudin disrupts higher order structures and channels formed by a different claudin, represents a previously unrecognized mechanism of barrier regulation.

Engineering and monitoring cellular barrier models.

Epithelia and endothelia delineate tissue compartments and control their environments by regulating the passage of ions and solutes. This barrier function is essential for the development and maintenance of multicellular organisms, and its dysfunction is associated with numerous human diseases. Recent advances in biomaterials and microfabrication technologies have evolved in vitro approaches for modelling biological barriers. Current microphysiological systems have become more efficient and reliable in mimicking the cell microenvironment. Additionally, methods for the quantification of barrier permeability have long provided significant insight into their underlying mechanisms. In this review, we outline the current techniques to quantify the barrier function of engineered tissues, and we also give an overview of recent microphysiological systems of biological barriers that emulate the microenvironment and microarchitecture of native tissues.

A perfusion chamber for monitoring transepithelial NaCl transport in an in vitro model of the renal tubule.

Transepithelial electrical measurements in the renal tubule have provided a better understanding of how kidney regulates electrolyte and water homeostasis through the reabsorption of molecules and ions (e.g., H2 O and NaCl). While experiments and measurement techniques using native tissue are difficult to prepare and to reproduce, cell cultures conducted largely with the Ussing chamber lack the effect of fluid shear stress which is a key physiological stimulus in the renal tubule. To overcome these limitations, we present a modular perfusion chamber for long-term culture of renal epithelial cells under flow that allows the continuous and simultaneous monitoring of both transepithelial electrical parameters and transepithelial NaCl transport. The latter is obtained from electrical conductivity measurements since Na+ and Cl- are the ions that contribute most to the electrical conductivity of a standard physiological solution. The system was validated with epithelial monolayers of raTAL and NRK-52E cells that were characterized electrophysiologically for 5 days under different flow conditions (i.e., apical perfusion, basal, or both). In addition, apical to basal chemical gradients of NaCl (140/70 and 70/140 mM) were imposed in order to demonstrate the feasibility of this methodology for quantifying and monitoring in real time the transepithelial reabsorption of NaCl, which is a primary function of the renal tubule.

Characterization of an encapsulated insulin secreting human pancreatic beta cell line in a modular microfluidic device.

Type I diabetes mellitus is characterised by the destruction of the insulin producing beta cells within the pancreas by the immune system. After the success of Edmonton protocol, islet transplantation has shown to be a promising therapy, but with the Achilles´ heel of the need of using immunosuppressive drugs. Currently, cell encapsulation technology represents a real alternative to protect transplanted islets from the host´s immune attack. Although preliminary in vitro studies with encapsulated cells have been traditionally performed under static conditions in terms of viability and efficiency, these static cultures do not represent a close approach to in vivo environments. We have developed and characterised different alginate-poly-l-lysine-alginate (APA) microcapsules loaded with the insulin producing 1.1B4 cell line. Static in vitro studies confirmed a constant insulin secretion and a boost of the secretion when the medium was enriched with glucose. Nevertheless, these results were not completely reproduced in a dynamic system by APA liquefied microcapsules containing 1.1B4 cells. The dynamic culture setting created by a microfluidic device, allowed the determination of the glucose response in APA liquefied microcapsules, showing that dynamic conditions can mimic better physiological in vivo environments.

A compartmentalized microfluidic chip with crisscross microgrooves and electrophysiological electrodes for modeling the blood-retinal barrier.

The interconnection of different tissue-tissue interfaces may extend organ-on-chips to a new generation of sophisticated models capable of recapitulating more complex organ-level functions. Single interfaces are largely recreated in organ-on-chips by culturing the cells on opposite sides of a porous membrane that splits a chamber in two or by connecting the cells of two adjacent compartments through microchannels. However, it is difficult to interconnect more than one interface using these approaches. To address this challenge, we present a novel microfluidic device where cells are arranged in parallel compartments and are highly interconnected through a grid of microgrooves, which facilitates paracrine signaling and heterotypic cell-cell contact between multiple tissues. In addition, the device includes electrodes on the substrate for the measurement of transepithelial electrical resistance (TEER). Unlike conventional methods for measuring the TEER where electrodes are on each side of the cell barrier, a method with only electrodes on the substrate has been validated. As a proof-of-concept, we have used the device to mimic the structure of the blood-retinal barrier by co-culturing primary human retinal endothelial cells (HREC), a human neuroblastoma cell line (SH-SY5Y), and a human retinal pigment epithelial cell line (ARPE-19). Cell barrier formations were assessed by a permeability assay, TEER measurements, and ZO-1 expression. These results validate the proposed microfluidic device with microgrooves as a promising in vitro tool for the compartmentalization and monitoring of barrier tissues.

A novel modular bioreactor to in vitro study the hepatic sinusoid.

We describe a unique, versatile bioreactor consisting of two plates and a modified commercial porous membrane suitable for in vitro analysis of the liver sinusoid. The modular bioreactor allows i) excellent control of the cell seeding process; ii) cell culture under controlled shear stress stimulus, and; iii) individual analysis of each cell type upon completion of the experiment. The advantages of the bioreactor detailed here are derived from the modification of a commercial porous membrane with an elastomeric wall specifically moulded in order to define the cell culture area, to act as a gasket that will fit into the bioreactor, and to provide improved mechanical robustness. The device presented herein has been designed to simulate the in vivo organization of a liver sinusoid and tested by co-culturing endothelial cells (EC) and hepatic stellate cells (HSC). The results show both an optimal morphology of the endothelial cells as well as an improvement in the phenotype of stellate cells, most probably due to paracrine factors released from endothelial cells. This device is proposed as a versatile, easy-to-use co-culture system that can be applied to biomedical research of vascular systems, including the liver.