Color tunable pressure sensors based on polymer nanostructured membranes for optofluidic applications

We demonstrate an integrated optical pressure sensing platform for multiplexed optofluidics applications. The sensing platform consists in an array of elastomeric on-side nanostructured membranes -effectively 2D photonic crystal- which present colour shifts in response to mechanical stress that alter their nanostructure characteristical dimensions, pitch or orientation. The photonic membranes are prepared by a simple and cost-effective method based on the infiltration of a 2D colloidal photonic crystal (CPC) with PDMS and their integration with a microfluidic system. We explore the changes in the white light diffraction produced by the nanostructured membranes when varying the pneumatic pressure in the microfluidics channels as a way to achieve a power-free array of pressure sensors that change their reflective colour depending on the bending produced on each sensor. The structural characterization of these membranes was performed by SEM, while the optical properties and the pressure-colour relation were evaluated via UV-Vis reflection spectrometry. Maximum sensitivities of 0.17 kPa is obtained when measuring at Littrow configuration (θ = −θ ), and close to the border of the membranes. The reflected colour change with pressure is as well monitorized by using a smartphone camera

Integrated Bioluminescent Immunoassays for High-Throughput Sampling and Continuous Monitoring of Cytokines

Immunoassays show great potential for the detection of low levels of cytokines, due to their high sensitivity and excellent specificity. There is a particular demand for biosensors that enable both high-throughput screening and continuous monitoring of clinically relevant cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα). To this end, we here introduce a novel bioluminescent immunoassay based on the ratiometric plug-and-play immunodiagnostics (RAPPID) platform, with an improved intrinsic signal-to-background and an &gt;80-fold increase in the luminescent signal. The new dRAPPID assay, comprising a dimeric protein G adapter connected via a semiflexible linker, was applied to detect the secretion of IL-6 by breast carcinoma cells upon TNFα stimulation and the production of low concentrations of IL-6 (∼18 pM) in an endotoxin-stimulated human 3D muscle tissue model. Moreover, we integrated the dRAPPID assay in a newly developed microfluidic device for the simultaneous and continuous monitoring of changes in IL-6 and TNFα in the low-nanomolar range. The luminescence-based read-out and the homogeneous nature of the dRAPPID platform allowed for detection with a simple measurement setup, consisting of a digital camera and a light-sealed box. This permits the usage of the continuous dRAPPID monitoring chip at the point of need, without the requirement for complex or expensive detection techniques.</p

Microphysiological systems for modelling and monitoring biological barriers /

Premi Extraordinari de Doctorat concedit pels programes de doctorat de la UAB per curs acadèmic 2017-2018Los sistemas microfisiológicos (MPS) son modelos in vitro microfabricados que emulan las condiciones in vivo fisiológicamente relevantes, como la organización celular y las señales microambientales. Las microtecnologías han permitido el desarrollo de sofisticados MPS capaces de recapitular fielmente la fisiología a nivel de tejido y órgano. Los MPS son particularmente útiles para modelar barreras biológicas, es decir, epitelios y endotelios que separan la circulación sanguínea de los compartimentos tisulares. Su función de barrera es crucial para mantener la homeostasis en los órganos y su desregulación juega un papel importante en la fisiopatología de muchas enfermedades humanas prevalentes. La función principal de un tejido barrera es controlar el transporte transepitelial de solutos. Por lo tanto, la capacidad de cuantificar el transporte en un modelo de barrera es crítico. La espectroscopía de impedancia eléctrica (EIS) permite su cuantificación con las ventajas de ser no destructiva, sin utilizar marcadores y de fácil aplicación en tiempo real. EIS puede determinar 1) la resistencia eléctrica transepitelial (TEER), que evalúa la integridad de la barrera (estrechamente relacionada con la rigidez del espacio intercelular); 2) la capacitancia de la capa celular (Ccl), que puede proporcionar información sobre el área superficial de la membrana; y 3) la contribución de la solución del medio a la impedancia. Mientras que el EIS es fácil de realizar mediante electrodos extracelulares, es difícil lograr la distribución de corriente uniforme requerida para mediciones precisas dentro de los canales de cultivo celular miniaturizados. Entonces, se puede suponer erróneamente que todo el área de cultivo de células contribuye igual a la medición, lo que puede conducir a errores de cálculo del TEER. Esto puede explicar parcialmente la gran disparidad de los valores de TEER reportados para tipos de células idénticas. Aquí, se presenta un estudio numérico para dilucidar este problema en algunos cultivos celulares previamente reportados y para proponer un factor de corrección geométrica (GCF) que corrige este error y que permite aplicarse retrospectivamente. Este estudio también se usó para optimizar una configuración tetrapolar especialmente adecuada para realizar mediciones EIS precisas en canales microfluídicos, y lo que es más importante, los electrodos cubren mínimamente la superficie lo que permite que las células se puedan visualizar junto con el análisis de TEER. Posteriormente, se desarrolló una cámara de perfusión modular con electrodos integrados en base a esta configuración óptima. El dispositivo comprende una membrana porosa desechable en la que se forma el tejido barrera y dos placas reutilizables donde se encuentran los electrodos. Por lo tanto, el tejido en la membrana se puede ensamblar en el sistema para medirlo y exponerlo al flujo, no solo para aplicar un estímulo mecánico fluidico sino también para suministrar continuamente nutrientes y eliminar los desechos. Además, la concentración de NaCl en ambos lados del tejido se puede estimar a partir de la conductancia eléctrica medida con los mismos electrodos integrados en una configuración bipolar. Un modelo in vitro del túbulo renal se utilizó para validar el sistema de medición. Como resultado, la concentración de NaCl se estimó a partir de la conductancia que permite la medición en línea del gradiente químico transepitelial de NaCl, que es una función primaria del túbulo renal. El desarrollo de MPS con múltiples barreras biológicas interconectadas expandirá la tecnología para recapitular funciones más complejas a nivel de órgano. Sin embargo, existen múltiples desafíos técnicos para reproducir varias barreras biológicas en un solo dispositivo mientras se mantiene un microambiente controlado particular para cada tipo de célula. Aquí se presenta un novedoso dispositivo microfluídico donde 1) múltiples tipos de células que están dispuestas en compartimentos uno al lado del otro están interconectadas con microsurcos y donde 2) múltiples tejidos barrera se miden a través de electrodos metálicos que están enterrados debajo de los microsurcos. Como prueba de concepto, el dispositivo se usó para imitar la estructura de la barrera hematorretiniana (BRB), incluidas las barreras interna y externa. Ambas barreras se formaron con éxito en el dispositivo y se monitorearon en tiempo real, lo que demuestra su gran potencial para su aplicación a la tecnología de órgano en un chipMicrophysiological systems (MPS) are biologically inspired microengineered in vitro models that emulate physiologically relevant in vivo conditions, such as cell organization and microenvironmental cues. Microtechnologies have enabled the development of significant MPS that are able to faithfully recapitulate tissue- and organ-level physiology. MPS are particularly useful for modelling biological barriers, that is, epithelia and endothelia that separate the blood circulation from tissue compartments. Their barrier function is crucial to maintain organ homeostasis and their deregulation play an important role in the pathophysiology of many prevalent human diseases. The primary function of a barrier tissue is to control the transepithelial transport of solutes. Therefore, the ability to quantify transport in a barrier model is critical. Electrical impedance spectroscopy (EIS) permits its quantification with the advantages of being non-destructive, label-free, and easily applicable in real time. EIS can determine 1) the transepithelial electrical resistance (TEER), which evaluates the barrier integrity (closely related with the tightness of the intercellular space); 2) the cell layer capacitance (Ccl), which can yield information about the membrane surface area; and 3) the contribution of the medium solution to the impedance. While EIS is easy to carry out by means of extracellular electrodes, it is challenging to achieve the uniform current distribution required for accurate measurements within miniaturized cell culture channels. Then, it may be erroneously assumed that the entire cell culture area contributes equally to the measurement leading to TEER calculation errors. This can partially explain the large disparity of TEER values reported for identical cell types. Here, a numerical study is presented to elucidate this issue in some cell cultures previously reported and to propose a geometric correction factor (GCF) to correct this error and be applied retrospectively. This study was also used to optimize a tetrapolar configuration especially suitable for performing accurate EIS measurements in microfluidic channels; importantly, it implements minimal electrode coverage so that the cells can be visualised alongside TEER analysis. A modular perfusion chamber with integrated electrodes was developed based on this optimal configuration. The device comprises a disposable porous membrane where the barrier tissue is formed and two reusable plates where the electrodes are located. Therefore, the tissue on the membrane can be assembled into the system to be measured and exposed to flow-not only to apply a fluid mechanical stimuli but also to continuously supply nutrients and remove waste. Additionally, the concentration of NaCl in both sides of the tissue can be estimated from the electrical conductance measured with the same integrated electrodes in a bipolar configuration. An in vitro model of the renal tubule was used to validate the measurement system. As a result, the concentration of NaCl was estimated from the conductance enabling in-line measurement of the transepithelial chemical gradient of NaCl, which is a primary function of the renal tubule. The development of MPS with multiple interconnected biological barriers will expand the technology to recapitulate more complex organ-level functions. Unfortunately, there are multiple technical challenges to reproduce several biological barriers in a single device while maintaining a particular controlled microenvironment for each cell type. Here, it is presented a novel microfluidic device where 1) multiple cell types that are arranged in side-by-side compartments are interconnected with microgrooves and where 2) multiple barrier tissues are measured through metal electrodes that are buried under the microgrooves. As a proof-of-concept, the device was used to mimic the structure of the blood-retinal barrier (BRB) including the inner and the outer barriers. Both barriers were successfully formed in the device and monitored in real time, demonstrating its great potential for application to organ-on-achip technology

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.This work has been supported by grants from the Ministerio de Economía y Competitividad (MINECO) (SAF2014–62114-EXP, DPI2015–65401-C3–3-R, and RYC-2013-14479) and from CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN).Peer reviewe

Device for measuring the trans-layer electrical impedance in an in vitro model of a cell barrier

A device mountable in an in vitro model of a cell barrier, the model comprising a first chamber, a second chamber and at least one support for separating the first chamber and the second chamber. The device comprises a first set of electrodes, a second set of electrodes, an element for connecting electrically the device to an electrical apparatus, the element comprising a first joint connected to the first set of electrodes and a second joint connected to the second set of electrodes. The first set of electrodes comprises a first electrode and a second electrode, said electrodes being adapted to be arranged in an interdigitated manner on an inner surface of the first chamber. The second set of electrodes comprises a first electrode and a second electrode, said electrodes being adapted to be arranged in an interdigitated manner on an inner surface of the second chamber.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Centro de Investigación Biomédica en RedA1 Solicitud de patente con informe sobre el estado de la técnic

Color tunable pressure sensors based on polymer nanostructured membranes for optofluidic applications

We demonstrate an integrated optical pressure sensing platform for multiplexed optofluidics applications. The sensing platform consists in an array of elastomeric on-side nanostructured membranes -effectively 2D photonic crystal- which present colour shifts in response to mechanical stress that alter their nanostructure characteristical dimensions, pitch or orientation. The photonic membranes are prepared by a simple and cost-effective method based on the infiltration of a 2D colloidal photonic crystal (CPC) with PDMS and their integration with a microfluidic system. We explore the changes in the white light diffraction produced by the nanostructured membranes when varying the pneumatic pressure in the microfluidics channels as a way to achieve a power-free array of pressure sensors that change their reflective colour depending on the bending produced on each sensor. The structural characterization of these membranes was performed by SEM, while the optical properties and the pressure-colour relation were evaluated via UV-Vis reflection spectrometry. Maximum sensitivities of 0.17 kPa is obtained when measuring at Littrow configuration (θ = −θ ), and close to the border of the membranes. The reflected colour change with pressure is as well monitorized by using a smartphone camera

Geometric correction factor for transepithelial electrical resistance measurements in transwell and microfluidic cell cultures

Transepithelial electrical resistance (TEER) measurements are regularly used in in vitro models to quantitatively evaluate the cell barrier function. Although it would be expected that TEER values obtained with the same cell type and experimental setup were comparable, values reported in the literature show a large dispersion for unclear reasons. This work highlights a possible error in a widely used formula to calculate the TEER, in which it may be erroneously assumed that the entire cell culture area contributes equally to the measurement. In this study, we have numerically calculated this error in some cell cultures previously reported. In particular, we evidence that some TEER measurements resulted in errors when measuring low TEERs, especially when using Transwell inserts 12 mm in diameter or microfluidic systems that have small chamber heights. To correct this error, we propose the use of a geometric correction factor (GCF) for calculating the TEER. In addition, we describe a simple method to determine the GCF of a particular measurement system, so that it can be applied retrospectively. We have also experimentally validated an interdigitated electrodes (IDE) configuration where the entire cell culture area contributes equally to the measurement, and it also implements minimal electrode coverage so that the cells can be visualized alongside TEER analysis.This work is part of the requirements to achieve the PhD degree in Electrical and Telecommunication Engineering at the Universitat Autònoma de Barcelona and it was supported by grants from CIBER-BBN, CSIC (PIE-201450E116) and Ministerio de Economía y Competitividad (SAF2014-62114-EXP and DPI2015-65401-C3-3-R). CIBER-BBN is funded by Instituto de Salud Carlos III. We are grateful to Drs. Pierre-Olivier Couraud (INSERM, France), Babette Weksler (Weill Cornell Medical College, New York, NY) and Ignacio Romero (Open University, Milton Keynes, UK) for kindly providing the hCMEC/D3 cell line. We would also acknowledge to Dr Mercedes Unzeta (Universitat Autònoma de Barcelona, Spain) for her advice and providing materials to perform the experiments with the cells.Peer reviewe

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.This work was supported by: CSIC (PIE 201450E116) (RV); Instituto de Salud Carlos III (FIS PI11/00235) (JGS); Ministerio de Economía y Competitividad (SAF2012-31238) (CP); European Union (Fondos FEDER, ‘‘una manera de hacer Europa’’) (JGS and CP); European Community’s Seventh Framework Programme(ECFP7/2007-2013, grant agreement 229673) (JGS); CIBER-EHD (funded by Instituto de Salud Carlos III) (JGS and CP); and CIBER-BBN (funded by Instituto de Salud Carlos III) (RV). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

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.This work is part of the requirements to achieve the PhD degree in Electrical and Telecommunication Engineering at the Universitat Autònoma de Barcelona, and it was supported by grants from Ministerio de Economía y Competitividad (MINECO) (SAF2014-62114-EXP and SAF2016-77784-R) and Fundació La Marató de TV3 (Exp. 201629-10). This work has made use of the Spanish ICTS Network MICRONANOFABS partially supported by MINECO and the ICTS 'NANBIOSIS', more specifically by the Micro-Nano Technology Unit of the CIBER in Bioengineering, Biomaterials & Nanomedicne (CIBER-BBN) at the IMB-CNM.Peer reviewe