Microdevices for cancer stem cell culture as a predictive chemotherapeutic response platform.

Microfluidic platforms for clinical use are a promising translational strategy for cancer research specially for drug screening. Identifying cancer stem cells (CSC) using sphere culture techniques in microfluidic devices (MDs) showed to be better reproducing physiological responses than other in vitro models and allow the optimization of samples and reagents. We evaluated individual sphere proliferation and stemness toward chemotherapeutic treatment (CT) with doxorubicin and cisplatin in bladder cancer cell lines (MB49-I and J82) cultured in MDs used as CSC treatment response platform. Our results confirm the usefulness of this device to evaluate the CT effect in sphere-forming efficiency, size, and growth rate from individual spheres within MDs and robust information comparable to conventional culture plates was obtained. The expression of pluripotency genetic markers (Oct4, Sox2, Nanog, and CD44) could be analyzed by qPCR and immunofluorescence in spheres growing directly in MDs. MDs are a suitable platform for sphere isolation from tumor samples and can provide information about CT response. Microfluidic-based CSC studies could provide information about treatment response of cancer patients from small samples and can be a promising tool for CSC-targeted specific treatment with potential in precision medicine. KEY MESSAGES: We have designed a microfluidic platform for CSC enriched culture by tumor sphere formation. Using MDs, we could quantify and determine sphere response after CT using murine and human cell lines as a proof of concept. MDs can be used as a tumor-derived sphere isolation platform to test the effect of antitumoral compounds in sphere proliferation.

Large Area Microfluidic Bioreactor for Production of Recombinant Protein.

To produce innovative biopharmaceuticals, highly flexible, adaptable, robust, and affordable bioprocess platforms for bioreactors are essential. In this article, we describe the development of a large-area microfluidic bioreactor (LM bioreactor) for mammalian cell culture that works at laminar flow and perfusion conditions. The 184 cm2 32 cisterns LM bioreactor is the largest polydimethylsiloxane (PDMS) microfluidic device fabricated by photopolymer flexographic master mold methodology, reaching a final volume of 2.8 mL. The LM bioreactor was connected to a syringe pump system for culture media perfusion, and the cells' culture was monitored by photomicrograph imaging. CHO-ahIFN-α2b adherent cell line expressing the anti-hIFN-a2b recombinant scFv-Fc monoclonal antibody (mAb) for the treatment of systemic lupus erythematosus were cultured on the LM bioreactor. Cell culture and mAb production in the LM bioreactor could be sustained for 18 days. Moreover, the anti-hIFN-a2b produced in the LM bioreactor showed higher affinity and neutralizing antiproliferative activity compared to those mAbs produced in the control condition. We demonstrate for the first-time, a large area microfluidic bioreactor for mammalian cell culture that enables a controlled microenvironment suitable for the development of high-quality biologics with potential for therapeutic use.

Bioremediation on a chip: A portable microfluidic device for efficient screening of bacterial biofilm with polycyclic aromatic hydrocarbon removal capacity.

Polycyclic aromatic hydrocarbons (PAHs) are pollutants of critical environmental and public health concern and their elimination from contaminated sites is significant for the environment. Biodegradation studies have demonstrated the ability of bacteria in biofilm conformation to enhance the biodegradation of pollutants. In this study, we used our newly developed microfluidic platform to explore biofilm development, properties, and applications of fluid flow, as a new technique for screening PAHs-degrading biofilms. The optimization and evaluation of the flow condition in the microchannels were performed through computational fluid dynamics (CFD). The formation of biofilms by PAHs-degrading bacteria Pseudomonas sp. P26 and Gordonia sp. H19, as pure cultures and co-culture, was obtained in the developed microchips. The removal efficiencies of acenaphthene, fluoranthene and pyrene were determined by HPLC. All the biofilms formed in the microchips removed all tested PAHs, with the higher removal percentages observed with the Pseudomonas sp. P26 biofilm (57.4% of acenaphthene, 40.9% of fluoranthene, and 28.9% of pyrene). Pseudomonas sp. P26 biofilm removed these compounds more efficiently than planktonic cultures. This work proved that the conformation of biofilms enhances the removal rate. It also provided a new tool to rapid and low-cost screen for effective pollutant-degrading biofilms.

A Reconfigurable In Vitro Model for Studying the Blood-Brain Barrier.

Much of what is currently known about the role of the blood-brain barrier (BBB) in regulating the passage of chemicals from the blood stream to the central nervous system (CNS) comes from animal in vivo models (requiring extrapolation to human relevance) and 2D static in vitro systems, which fail to capture the rich cell-cell and cell-matrix interactions of the dynamic 3D in vivo tissue microenvironment. In this work we have developed a BBB platform that allows for a high degree of customization in cellular composition, cellular orientation, and physiologically-relevant fluid dynamics. The system characterized and presented in this study reproduces key characteristics of a BBB model (e.g. tight junctions, efflux pumps) allowing for the formation of a selective and functional barrier. We demonstrate that our in vitro BBB is responsive to both biochemical and mechanical cues. This model further allows for culture of a CNS-like space around the BBB. The design of this platform is a valuable tool for studying BBB function as well as for screening of novel therapeutics.

Hybrid microchannel-solid state micropore device for fast and optical cell detection.

This paper presents a methodology for cell detection and counting using a device that combines PDMS (polydimethylsiloxane) microfluidic multilayer channels with a single solid state micropore. Optimal conditions of solid-state micropore fabrication from crystalline silicon wafers are presented. Micropores of varying size can be obtained by directly etching using an etchant agent concentration of 50 wt% KOH, at varying temperatures (40, 60, 80 °C) and voltages (100, 500, 1000 mV). Scanning Electron Microscopy (SEM), and profilometry techniques have been used for the micropore characterization. In order to find optimal conditions for cell detection a COMSOL Multiphysics simulation was performed. Pressure drop, shear stress, fluid viscosities and flow rates parameters were evaluated. The potential viability of the device for cell detection and counting, avoiding cellular damage, is demonstrated.

Analysis of tumoral spheres growing in a multichamber microfluidic device.

Lab on a Chip (LOC) farming systems have emerged as a powerful tool for single cell studies combined with a non-adherent cell culture substrate and single cell capture chips for the study of single cell derived tumor spheres. Cancer is characterized by its cellular heterogeneity where only a small population of cancer stem cells (CSCs) are responsible for tumor metastases and recurrences. Thus, the in vitro strategy to the formation of a single cell-derived sphere is an attractive alternative to identify CSCs. In this study, we test the effectiveness of microdevices for analysis of heterogeneity within CSC populations and its interaction with different components of the extracellular matrix. CSC could be identify using specific markers related to its pluripotency and self-renewal characteristics such as the transcription factor Oct-4 or the surface protein CD44. The results confirm the usefulness of LOC as an effective method for quantification of CSC, through the formation of spheres under conditions of low adhesion or growing on components of the extracellular matrix. The device used is also a good alternative for evaluating the individual growth of each sphere and further identification of these CSC markers by immunofluorescence. In conclusion, LOC devices have not only the already known advantages, but they are also a promising tool since they use small amounts of reagents and are under specific culture parameters. LOC devices could be considered as a novel technology to be used as a complement or replacement of traditional studies on culture plates.

Production of monoclonal antibodies in microfluidic devices.

Herein, a microfluidic device with cistern design for cultivation of adherent eukaryotic cells for the production of recombinant proteins is presented. The geometric configuration of the microchannels in the device provided laminar flow with reduced velocity profiles in the cisterns, resulting in an adequate microenvironment for long-term adherent cell growth with passive pumping flow cycles of 24 hours. CHO-ahIFNα2b and HEK-ahIFNα2b adherent cell lines expressing a novel anti-hIFN-α2b recombinant monoclonal antibody (MAb) for the treatment of systemic lupus erythematosus were cultured on the surface of PDMS/glass microchannels coated with poly-d-lysine. A 24 day culture of CHO-ahIFNα2b cells resulted in MAb concentrations up to 166.4 µg mL-1 per day. The productivity of CHO-ahIFNα2b and HEK-ahIFNα2b cell lines was higher in the microdevice compared to that obtained using the adherent cell culture method (T-flask), with a 5.89- and 7.31-fold increase, respectively. Moreover, biological analysis of the MAbs produced in the microdevice showed no significant differences in the neutralizing antiproliferative activity of the hIFN-α2b or the cytokine cell signaling compared to the MAbs produced with cell adherent methods. These results suggest that this microfluidic device is suitable for long-term culture of mammalian cells and can improve the productivity of cells expressing recombinant MAbs with potential for therapeutic use without affecting the quality attributes of the product.

Simultaneous electrical recording of cardiac electrophysiology and contraction on chip.

Prevailing commercialized cardiac platforms for in vitro drug development utilize planar microelectrode arrays to map action potentials, or impedance sensing to record contraction in real time, but cannot record both functions on the same chip with high spatial resolution. Here we report a novel cardiac platform that can record cardiac tissue adhesion, electrophysiology, and contractility on the same chip. The platform integrates two independent yet interpenetrating sensor arrays: a microelectrode array for field potential readouts and an interdigitated electrode array for impedance readouts. Together, these arrays provide real-time, non-invasive data acquisition of both cardiac electrophysiology and contractility under physiological conditions and under drug stimuli. Human induced pluripotent stem cell-derived cardiomyocytes were cultured as a model system, and used to validate the platform with an excitation-contraction decoupling chemical. Preliminary data using the platform to investigate the effect of the drug norepinephrine are combined with computational efforts. This platform provides a quantitative and predictive assay system that can potentially be used for comprehensive assessment of cardiac toxicity earlier in the drug discovery process.

Characterization of a microfluidic in vitro model of the blood-brain barrier (µBBB).

The blood-brain barrier (BBB), a unique selective barrier for the central nervous system (CNS), hinders the passage of most compounds to the CNS, complicating drug development. Innovative in vitro models of the BBB can provide useful insights into its role in CNS disease progression and drug delivery. Static transwell models lack fluidic shear stress, while the conventional dynamic in vitro BBB lacks a thin dual cell layer interface. To address both limitations, we developed a microfluidic blood-brain barrier (µBBB) which closely mimics the in vivo BBB with a dynamic environment and a comparatively thin culture membrane (10 µm). To test validity of the fabricated BBB model, µBBBs were cultured with b.End3 endothelial cells, both with and without co-cultured C8-D1A astrocytes, and their key properties were tested with optical imaging, trans-endothelial electrical resistance (TEER), and permeability assays. The resultant imaging of ZO-1 revealed clearly expressed tight junctions in b.End3 cells, Live/Dead assays indicated high cell viability, and astrocytic morphology of C8-D1A cells were confirmed by ESEM and GFAP immunostains. By day 3 of endothelial culture, TEER levels typically exceeded 250 Ω cm(2) in µBBB co-cultures, and 25 Ω cm(2) for transwell co-cultures. Instantaneous transient drop in TEER in response to histamine exposure was observed in real-time, followed by recovery, implying stability of the fabricated µBBB model. Resultant permeability coefficients were comparable to previous BBB models, and were significantly increased at higher pH (>10). These results demonstrate that the developed µBBB system is a valid model for some studies of BBB function and drug delivery.