Within the tumor microenvironment (TME), cancer cells use mechanotransduction pathways to convert biophysical forces to biochemical signals. However, the underlying mechanisms and functional... Show moreWithin the tumor microenvironment (TME), cancer cells use mechanotransduction pathways to convert biophysical forces to biochemical signals. However, the underlying mechanisms and functional significance of these pathways remain largely unclear. The upregulation of mechanosensitive pathways from biophysical forces such as interstitial flow (IF), leads to the activation of various cytokines, including transforming growth factor-β (TGF-β). TGF-β promotes in part via a Smad-dependent signaling pathway the epithelial–mesenchymal transition (EMT) in cancer cells. The latter process is linked to increased cancer cell motility and invasion. Current research models have limited ability to investigate the combined effects of biophysical forces (such as IF) and cytokines (TGF-β) in a 3D microenvironment. We used a 3D-matrix based microfluidic platform to demonstrate the potentiating effect of IF on exogenous TGF-β induced upregulation of the Smad-signaling activity and the expression of mesenchymal marker vimentin in A549 lung cancer spheroids. To monitor this, we used stably integrated fluorescent based reporters into the A549 cancer cell genome. Our results demonstrate that IF enhances exogenous TGF-β induced Smad-signaling activity in lung cancer spheroids embedded in a matrix microenvironment. In addition, we observed an increased cell motility for A549 spheroids when exposed to IF and TGF-β. Our 3D-microfluidic model integrated with real-time imaging provides a powerful tool for investigating cancer cell signaling and motility associated with invasion characteristics in a physiologically relevant TME. Show less
Graaf, M.N.S. de; Vivas, A.; Kasi, D.G.; Hil, F.E. van den; Berg, A. van den; Meer, A.D. van der; ... ; Orlova, V.V. 2022
Three-dimensional (3D) blood vessels-on-a-chip (VoC) models integrate the biological complexity of vessel walls with dynamic microenvironmental cues, such as wall shear stress (WSS) and... Show moreThree-dimensional (3D) blood vessels-on-a-chip (VoC) models integrate the biological complexity of vessel walls with dynamic microenvironmental cues, such as wall shear stress (WSS) and circumferential strain (CS). However, these parameters are difficult to control and are often poorly reproducible due to the high intrinsic diameter variation of individual 3D-VoCs. As a result, the throughput of current 3D systems is one-channel-at-a-time. Here, we developed a fluidic circuit board (FCB) for simultaneous perfusion of up to twelve 3D-VoCs using a single set of control parameters. By designing the internal hydraulic resistances in the FCB appropriately, it was possible to provide a pre-set WSS to all connected 3D-VoCs, despite significant variation in lumen diameters. Using this FCB, we found that variation of CS or WSS induce morphological changes to human induced pluripotent stem cell (hiPSC)-derived endothelial cells (ECs) and conclude that control of these parameters using a FCB is necessary to study 3D-VOCs. Show less
Organ-on-chip devices are intensively studied in academia and industry due to their high potential in pharmaceutical and biomedical applications. However, most of the existing organ-on-chip models... Show moreOrgan-on-chip devices are intensively studied in academia and industry due to their high potential in pharmaceutical and biomedical applications. However, most of the existing organ-on-chip models focus on proof of concept of individual functional units without the possibility of testing multiple experimental stimuli in parallel. Here we developed a polydimethylsiloxane (PDMS) multiplexed chip with eight parallel channels branching from a common access port through which all eight channels can be addressed simultaneously without the need for extra pipetting steps thus increasing the reproducibility of the experimental results. At the same time, eight outlets provide individual entry to each channel with the opportunity to create eight different experimental conditions. A multiplexed chip can be assembled as a one-layer device for studying monocultures or as a two-layer device for studying barrier tissue functions. For a two-layer device, a similar to 2 mu m thick transparent PDMS membrane with 5 mu m through-hole pores was fabricated in-house using a soft lithography technique, thereby allowing visual inspection of the cell-culture in real-time. The functionality of the chip was studied by recapitulating the blood-brain barrier. For this, human cerebral microvascular endothelial cells (hCMEC/D3) were cultured in mono- or coculture with human astrocytes. Immunostaining revealed a cellular monolayer with the expression of tight junction ZO-1 and adherence junction VE-cadherin proteins in endothelial cells as well as glial fibrillary acidic protein (GFAP) expression in astrocytes. Furthermore, multiplexed permeability studies of molecule passage through the cellular barrier exhibited expected high permeability coefficients for smaller molecules (4 kDa FITC-dextran) whereas larger molecules (20 kDa) crossed the barrier at a lower rate. With these results, we show that our device can be used as an organ-on-chip model for future multiplexed drug testing. Show less
Dijk, C.G.M. van; Brandt, M.M.; Poulis, N.; Anten, J.; Moolen, M. van der; Kramer, L.; ... ; Cheng, C. 2020
Microfluidic organ-on-a-chip designs are used to mimic human tissues, including the vasculature. Here we present a novel microfluidic device that allows the interaction of endothelial cells (ECs)... Show moreMicrofluidic organ-on-a-chip designs are used to mimic human tissues, including the vasculature. Here we present a novel microfluidic device that allows the interaction of endothelial cells (ECs) with pericytes and the extracellular matrix (ECM) in full bio-matrix encased 3D vessel structures (neovessels) that can be subjected to continuous, unidirectional flow and perfusion with circulating immune cells. We designed a polydimethylsiloxane (PDMS) device with a reservoir for a 3D fibrinogen gel with pericytes. Open channels were created for ECs to form a monolayer. Controlled, continuous, and unidirectional flow was introduced via a pump system while the design facilitated 3D confocal imaging. In this vessel-on-a-chip system, ECs interact with pericytes to create a human cell derived blood vessel which maintains a perfusable lumen for up to 7 days. Dextran diffusion verified endothelial barrier function while demonstrating the beneficial role of supporting pericytes. Increased permeability after thrombin stimulation showed the capacity of the neovessels to show natural vascular response. Perfusion of neovessels with circulating THP-1 cells demonstrated this system as a valuable platform for assessing interaction between the endothelium and immune cells in response to TNF alpha. In conclusion: we created a novel vascular microfluidic device that facilitates the fabrication of an array of parallel soft-channel structures in ECM gel that develop into biologically functional neovessels without hard-scaffold support. This model provides a unique tool to conduct live in vitro imaging of the human vasculature during perfusion with circulating cells to mimic (disease) environments in a highly systematic but freely configurable manner. Show less
Kramer, R.C.L.N.; Verlinden, E.J.; Angeloni, L.; Heuvel, A. van den; Fratila-Apachitei, L.E.; Maarel, S.M. van der; Ghatkesar, M.K. 2020
Microfluidic atomic force microscopy (AFM) cantilever probes have all the functionalities of a standard AFM cantilever along with fluid pipetting. They have a channel inside the cantilever and an... Show moreMicrofluidic atomic force microscopy (AFM) cantilever probes have all the functionalities of a standard AFM cantilever along with fluid pipetting. They have a channel inside the cantilever and an aperture at the tip. Such probes are useful for precise fluid manipulation at a desired location, for example near or inside cells. They are typically made by complex microfabrication process steps, resulting in expensive probes. Here, we used two different 3D additive manufacturing techniques, stereolithography and two-photon polymerization, to directly print ready-to-use microfluidic AFM cantilever probes. This approach has considerably reduced the fabrication time and increased the design freedom. One of the probes, 564 mu m long, 30 mu m wide, 30 mu m high, with a 25 mu m diameter channel and 2.5 mu m wall thickness had a spring constant of 3.7 N m(-1) and the polymer fabrication material had an elastic modulus of 4.2 GPa. Using these 3D printed probes, AFM imaging of a surface, puncturing of the cell membrane, and aspiration at the single cell level have been demonstrated. Show less
Brink, F.T.G. van den; Phisonkunkasem, T.; Asthana, A.; Bomer, J.G.; Maagdenberg, A.M.J.M. van den; Tolner, E.A.; Odijk, M. 2019
Measuring biomolecule concentrations in the brain of living animals, in real time, is a challenging task, especially when detailed information at high temporal resolution is also required.... Show moreMeasuring biomolecule concentrations in the brain of living animals, in real time, is a challenging task, especially when detailed information at high temporal resolution is also required. Traditionally, microdialysis probes are used that generally have sampling areas in the order of about 1 mm(2), and provide information on concentrations with a temporal resolution of at least several minutes. In this paper, we present a novel miniaturized push-pull perfusion sampling probe that uses an array of small 3 m-wide sampling channels to sample neurotransmitters at a typical recovery rate of 61%, with a reduced risk of clogging. The added feature to segment the dialysate inside the probe into small water-in-decane droplets enables the detection of concentrations with a temporal resolution of a few seconds. Here we used the probe for in vivo recordings of neurotransmitter glutamate released upon electrical stimulation in the brain of a mouse to demonstrate the feasibility of the probe for real-time neurochemical brain analysis. Show less
