Organ-on-chip (OoC) systems are novel microfluidic microsystems that combine the advantages of well-characterised human cells with the benefits of engineered, physiological-like microenvironments manufactured in the system. The extracellular matrix (ECM) is the natural microenvironment of cells in the human body responsible for providing the appropriate stimuli to cells to control cell processes such as proliferation, migration, and apoptosis. OoCs can mimic the ECM, via channels and porous membranes, by providing the cells with physiological-like mechanical stimuli governed by the fluid dynamics in the system [1]. Understanding the fluid dynamics in OOC can aid in fine-tuning the stimuli sensed by the cultured cells, understanding cell behavior and cell fate. The current state of the art methods for characterizing fluid dynamics in the OoC systems are simulations, theoretical calculations, and empirical observations, therefore a quantitative characterization technique is lacking. Optical coherence tomography (OCT) has been used in previous studies to measure omnidirectional flow velocities in flow systems [2]. In this study, we measured the flow in a cuvette using a Thorlabs GANYMEDE II HR series (high axial resolution of 3 mm in air) spectral domain OCT system. We made quantitative 2D flow measurements using the phase-resolved Doppler method. This work was then extended to extract flow dynamics, in the Bi/ond inCHIPit using titania scattering nanoparticles, which would be a novel way of flow characterization in the field of OOC. The results are compared to the theoretical Hagen-Poiseuille equations and COMSOL simulations and found to be in good agreement. The results of the study were further extended to determine the shear stress experienced by the cells in the culture well of the OoC. @en

The Smart Multi-Well Plate (SMWP), an open technology platform for Organ-on-Chip (OoC) technology developed as part of the Moore4Medical (M4M) consortium, aims to showcase the advantages of standardization in design, manufacturing and assembly for OoC [1]. In previously presented work [2], we showed integration and characterization of piezoelectric micropumps for in-line perfusion of OoC devices. Here we present the integration and preliminary biological evaluation of three OoC devices in a SMWP prototype. This prototype, a downscaled version of the full SMWP, is constructed using stacked, predefined layers. The reservoirs of a 96-well plate are fluidically connected to integrated OoC devices and micropumps through a fluidic circuit board (FCB). A printed circuit board, assembled below the FCB, enables the electrical interfacing. The following devices were integrated in the prototype: OoC devices from Bi/ond and BEOnChip, a microelectrode array (MEA) device from MultiChannel Systems, and piezoelectric micropumps from Fraunhofer EMFT. In the OoC devices, cell culture was performed on integrated on-chip membranes. On the MEA, neuronal cells were cultured directly on the surface of the chip. For initial experiments, static cultures were performed to investigate the biocompatibility of all included materials inside the prototype. BEOnChip performed a static cell culture with skin cells (Ha- CaT) in their device. Normal cell viability and morphology was observed after 96 h and 21 days of culture using Calcein-AM/PI live/ dead staining. MCS performed static cell culture using hiPSCs-derived neurons directly cultured on the PLO/laminin-coated MEA chips. After 14 days of culture, eGFP staining showed normal cell morphology and network formation. Bi/ond performed an endothelial cell culture (HMEC-1), showing proper cell adhesion and viability in their devices. The biological experiments under static conditions showed normal cell morphology and viability in all integrated devices. In the next phase of the project, the full SMWP platform with integrated perfusion will be used for biological experimentation to generate an air-liquid interface with skin cells (BEOnChip), a perfusable MEA (MCS) and endothelial tube formation under unidirectional flow (Bi/ond). @en

One of the many applications of organ-on-a-chip (OOC) technology is the study of biological processes in human induced pluripotent stem cells (iPSCs) during pharmacological drug screening. It is of paramount importance to construct OOCs equipped with highly compact in situ sensors that can accurately monitor, in real time, the extracellular fluid environment and anticipate any vital physiological changes of the culture. In this paper, we report the co-fabrication of a CMOS smart sensor on the same substrate as our silicon-based OOC for real-time in situ temperature measurement of the cell culture. The proposed CMOS circuit is developed to provide the first monolithically integrated in situ smart temperature-sensing system on a micromachined silicon-based OOC device. Measurement results on wafer reveal a resolution of less than ±0.2 °C and a nonlinearity error of less than 0.05% across a temperature range from 30 to 40 °C. The sensor's time response is more than 10 times faster than the time constant of the convection-cooling mechanism found for a medium containing 0.4 ml of PBS solution. All in all, this work is the first step towards realizing OOCs with seamless integrated CMOS-based sensors capable to measure, in real time, multiple physical quantities found in cell culture experiments. It is expected that the use of commercial foundry CMOS processes may enable OOCs with very large scale of multi-sensing integration and actuation in a closed-loop system manner.

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We present the first Organ-on-Chip equipped with a low-impedance Poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) MicroElectrode Array (MEA). The novel device allows simultaneous mechanical stimulation with a stretchable PDMS membrane and electrical monitoring via the PEDOT:PSS MEA of multiple in vitro cell cultures. The surface area enhancement and the morphology of the PEDOT:PSS allows an increase of the charge injection per unit area at the electrode-electrolyte interface, resulting in significantly lower electrochemical impedance of the electrodes. In particular, at 1 kHz the fabricated PEDOT-MEA electrodes show a reduction of the overall impedance up to 99.4 and 93.3 % in comparison with benchmark TiN and Pt electrodes. The superior performance of PEDOT:PSS were also confirmed via Cyclic Voltammetry measurement, in which PEDOT:PSS showed a very large capacitive current, compared with the benchmark electrodes both in the forward and the reverse scans. The obtained results confirm the effectiveness of the proposed PEDOT:PSS coating, and introduce this material in the OOC field. Moreover, the quality and morphology of the fabricated PEDOT:PSS based electrodes were assessed via SEM imaging and Raman spectroscopy.

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The cost and the development time of pharamecutical products are often severely affected by the in vitro tests currently employed in pharmaceutical R&D. These assays are often failing to accurately recapitulate diseases and to predict human responses to new medicines. Organ-on-Chip (OOC) devices are designed to result in advanced in vitro assays that better replicate human responses. The increasing interest and demand for this new class of devices is pushing for a quick commercialization of these tools. However, the currently employed fabrication processes pose major technical hurdles towards large-scale manufacturing, higher throughput and robustness, which are important steps for industrial adoption. This thesis aims to address these challenges, by implementing conventional cleanroom-compatible microfabrication processes for their fabrication. The aim is to copy, in the OOC field, what has been done in the microelectro- mechanical system (MEMS) field, where the standardization of surface patterning techniques using lithography and etching have been a major factor in their success. The OOCs resulting from this effort are named Organ-on-Silicon (OOS) devices in this thesis...@en

Organ-on-chip (OOC) is becoming the alternative tool to conventional in vitro screening. Heart-on-chip devices including microstructures for mechanical and electrical stimulation have been demonstrated to be advantageous to study structural organization and maturation of heart cells. This paper presents the development of metal and polymeric strain gauges for in situ monitoring of mechanical strain in the Cytostretch platform for heart-on-chip application. Specifically, the optimization of the fabrication process of metal titanium (Ti) strain gauges and the investigation on an alternative material to improve the robustness and performance of the devices are presented. The transduction behavior and functionality of the devices are successfully proven using a custom-made set-up. The devices showed resistance changes for the pressure range (0-3 kPa) used to stretch the membranes on which heart cells can be cultured. Relative resistance changes of approximately 0.008% and 1.2% for titanium and polymeric strain gauges are respectively reported for membrane deformations up to 5%. The results demonstrate that both conventional IC metals and polymeric materials can be implemented for sensing mechanical strain using robust microfabricated organ-on-chip devices.

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We present a novel and highly reproducible process to fabricate transferable porous PDMS membranes for PDMS-based Organs-on-Chips (OOCs) using microelectromechanical systems (MEMS) fabrication technologies. Porous PDMS membranes with pore sizes down to 2.0 μm in diameter and a wide porosity range (2–65%) can be fabricated. To overcome issues normally faced when using replica moulding and extend the applicability to most OOCs and improve their scalability and reproducibility, the process includes a sacrificial layer to easily transfer the membranes from a silicon carrier to any PDMS-based OOC. The highly reliable fabrication and transfer method does not need of manual handling to define the pore features (size, distribution), allowing very thin (<10 μm) functional membranes to be transferred at chip level with a high success rate (85%). The viability of cell culturing on the porous membranes was assessed by culturing two different cell types on transferred membranes in two different OOCs. Human umbilical endothelial cells (HUVEC) and MDA-MB-231 (MDA) cells were successfully cultured confirming the viability of cell culturing and the biocompatibility of the membranes. The results demonstrate the potential of controlling the porous membrane features to study cell mechanisms such as transmigrations, monolayer formation, and barrier function. The high control over the membrane characteristics might consequently allow to intentionally trigger or prevent certain cellular responses or mechanisms when studying human physiology and pathology using OOCs.

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This work presents the first multi-well plate that allows for simultaneous mechanical stimulation and electrical monitoring of multiple in-vitro cell cultures in parallel. Each well of the plate is equipped with an Organ-on-Chip (OOC) device consisting of a stretchable micro-electrode array (MEA). For the first time, a film assisted molding (FAM) process was employed to embed an OOC into a multi well plate format packaging. The functionality of the MEA in the device was assessed with electrochemical impedance spectroscopy. Moreover, the biocompatibility of the plate was demonstrated with cardiomyocytes derived from human induced pluripotent stem cells (iPSC) cultured in the wells.

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We present a novel method to easily and reliably transfer highly porous, large area, thin microfabricated Polydimethylsiloxane (PDMS) porous membranes on Lab-on-Chip (LOC) and Organ-on-Chip (OOC) devices. The use of silicon as carrier substrate and a water-soluble sacrificial layer allows a simple and reproducible transfer of the membranes to any PDMS-based OOC and LOC device. The use of IC and MEMS compatible techniques reduces significantly the fabrication time and the need of manual handling. Our method is suitable for automatic assembling systems, such as pick-and-place, crucial to significantly increase the throughput of OOC and LOC devices assembling. Membranes with 8 μm pore size and as thin as 4 μm are successfully transferred. The viability and biocompatibility of the transfer was assessed by culturing two different cell lines on an OOC with transferred porous PDMS membranes.

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Polymeric (PEDOT:PSS) strain gauges embedded in PDMS membranes fabricated using a full wafer-scale fabrication process capable of realizing reproducible small features, are reported. The devices are characterized using a customized setup, which provides mechanical stretch while dynamically reading the electrical resistance. Measurements show relative resistance changes of approximately 11% for applied pressure up to 4 kPa. The process described is tailored to fabricate pressure sensors and microelectrodes for a flexible substrate-based Organ-on-Chip platform.

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Organ-on-Chips (OOCs) are micro-fabricated devices which are used to culture cells in order to mimic functional units of human organs. The devices are designed to simulate the physiological environment of tissues in vivo. Cells in some types of OOCs can be stimulated in situ by electrical and/or mechanical actuators. These actuations can mimic physiological conditions in real tissue and may include fluid or air flow, or cyclic stretch and strain as they occur in the lung and heart. These conditions similarly affect cultured cells and may influence their ability to respond appropriately to physiological or pathological stimuli. To date, most focus has been on devices specifically designed to culture just one functional unit of a specific organ: lung alveoli, kidney nephrons or blood vessels, for example. In contrast, the modular Cytostretch membrane platform described here allows OOCs to be customized to different OOC applications. The platform utilizes silicon-based micro-fabrication techniques that allow low-cost, high-volume manufacturing. We describe the platform concept and its modules developed to date. Membrane variants include membranes with (i) through-membrane pores that allow biological signaling molecules to pass between two different tissue compartments; (ii) a stretchable micro-electrode array for electrical monitoring and stimulation; (iii) micro-patterning to promote cell alignment; and (iv) strain gauges to measure changes in substrate stress. This paper presents the fabrication and the proof of functionality for each module of the Cytostretch membrane. The assessment of each additional module demonstrate that a wide range of OOCs can be achieved.
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Sensing and stimulating microstructures are necessary to develop more specialized and highly accurate Organ-on-Chip (OOC) platforms. In this paper, we present the integration of a conductive polymer, poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), on a stretchable membrane, core element of an Heart-on-Chip. The electrical conductivity along with its biocompatibility, high transparency (≈88%) and mechanical elasticity (≈1.2 GPa) make this material a candidate to develop novel microstructures for electrical monitoring and stimulation of cells in flexible-substrate based OOCs. Microstructures with different shapes and geometries of PEDOT:PSS embedded in a 9 μm-thick Polydimethylsiloxane (PDMS) membrane are developed following a wafer-level fabrication approach. PEDOT:PSS layers between 120 nm and 300 nm are obtained by varying the deposition conditions. The layers are successfully patterned and microstructures with lateral dimensions down to 2 μm. The obtained results indicate that this polymer is a suitable material for microfabrication of sensing and stimulating elements in OOC platforms.

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Microelectrode arrays (MEAs) are widely used in biological application to locally stimulate and record the electrical activity of living cells. Here, a novel fabrication process for a carbon nanotube (CNT)-based MEA integrated on the backside of a free standing stretchable membrane is reported. The new process flow overcomes the manually intensive procedures used in the previous works. The microfabricated upside-down CNT MEA consists of microelectrodes with an area of 110 μm2 covered with cobalt-grown CNTs. The surface area enhancement and the foamlike morphology of the CNTs allow an increase of the charge injection per unit area at the electrode–electrolyte interface, resulting in a significantly lower electrochemical impedance of the electrodes. In particular, at 1 kHz, the fabricated CNT-MEA electrodes show a reduction of the overall impedance up to 96% in comparison with benchmark TiN electrodes. The obtained results confirm the effectiveness of the proposed surface texturing through CNT integration. Moreover, the quality and the morphology as well as the biocompatibility of the fabricated CNT-based electrodes were assessed. The obtained results demonstrate that significant improvement can be achieved by integrating structured nanoporous material on MEAs.@en