Drug development continuously faces challenges in efficiently predicting toxicity and verifying efficacy of new compounds during the early pre-clinical phases [1]. Organs-on-Chips (OOCs) have recently emerged as innovative in vitro tools holding the potential to improve prediction over human drug responses earlier in the timeline, thus reducing failures and costs of the clinical trials [2]. Here we present new beating OOCs providing cells with a 3D environment and mimicking the mechanical stimulation that tissues sense in vivo. This results in improved functionalities or induction of pathological changes. Our technology, named uBeat, relies on specific geometrical structures that modulate the mechanical deformation exerted on 3D microtissues, to achieve different magnitudes of either uniaxial strain or confined compression. Based on uBeat, we developed two platforms: i) uHeart, a spontaneously beating heart-on-chip integrating real-time measurement of cardiac electrophysiological signals [3] and ii) uKnee, the first in vitro 3D model of human osteoarthritic (OA) cartilage on chip [4]. We also exploited our models for drug screening purposes, by testing the effect of well-known compounds. uHeart provides 3D cardiac microtissues with a physiological cyclic uniaxial strain (~10%, 1Hz) and integrates electrodes to specifically measure the field potential (FP) signals online. Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) and human dermal fibroblast (h-DFs) were embedded in fibrin hydrogel in a 3:1 ratio and cultured for 7 days within uHeart. The cardiac model developed a synchronous beating and the electrical activity was recorded through paired electrodes specifically inserted. We preliminary calibrated the system by assessing the pro-arrhythmic effect of known compounds (i.e. Verapamil, Sotalol and Terfenadine) on uHeart electrical activity. Aspirin and DMSO were used as negative control and vehicle, respectively. As expected, Terfenadine and Sotalol prolonged the repolarization time of cardiac microtissues at 100-1000 nM and 10-60 µM, respectively. In contrast, Verapamil decreased the FP duration and both Aspirin (up to 100 µM) and DMSO (up to 0.5% w/v) did not alter the beating properties. uKnee provides 3D cartilage-like constructs with either physiological (10%, 1Hz), or hyper-physiological compression (30%,1 Hz). The latter is able to elicit OA pathogenesis by mechanical factors. Primary human articular chondrocytes were embedded in a poly(ethylene-glycol)-based hydrogel and cultured in uKnee under static chondrogenic conditions for 2 weeks. Deposition of a cartilage-like matrix was demonstrated by immunofluorescence staining (i.e. Aggrecan, Collagen II) and a stable cartilage phenotype was evidenced by the increased expression of specific genes (i.e. ACAN, PRG4, ATX, FRZB and GREM1). After maturation, constructs were subjected to additional 7 days of confined compression at both intensity levels. Hyper-physiological compression induced OA-like traits and significantly enhanced catabolic and inflammatory response, as evidenced by MMP13 and IL8 gene expression. Responses to 4 drugs already used in the clinic (i.e. Rapamacyn, Celecoxib, IL-1Ra and dexamethasone) were assessed and resulted consistent with data from animal studies, probing the potential of uKnee as anti-OA drugs screening platform. Our new technology (uBeat) allows to develop beating OOC as powerful and reliable pre-clinical tools for efficient in vitro drug screening and disease modelling.

Beating organs-on-chip as advanced tools in drug screening: Engineered in vitro models of human organs and diseases

Roberta Visone;Andrea Mainardi;Paola Occhetta;Marco Rasponi
2019

Abstract

Drug development continuously faces challenges in efficiently predicting toxicity and verifying efficacy of new compounds during the early pre-clinical phases [1]. Organs-on-Chips (OOCs) have recently emerged as innovative in vitro tools holding the potential to improve prediction over human drug responses earlier in the timeline, thus reducing failures and costs of the clinical trials [2]. Here we present new beating OOCs providing cells with a 3D environment and mimicking the mechanical stimulation that tissues sense in vivo. This results in improved functionalities or induction of pathological changes. Our technology, named uBeat, relies on specific geometrical structures that modulate the mechanical deformation exerted on 3D microtissues, to achieve different magnitudes of either uniaxial strain or confined compression. Based on uBeat, we developed two platforms: i) uHeart, a spontaneously beating heart-on-chip integrating real-time measurement of cardiac electrophysiological signals [3] and ii) uKnee, the first in vitro 3D model of human osteoarthritic (OA) cartilage on chip [4]. We also exploited our models for drug screening purposes, by testing the effect of well-known compounds. uHeart provides 3D cardiac microtissues with a physiological cyclic uniaxial strain (~10%, 1Hz) and integrates electrodes to specifically measure the field potential (FP) signals online. Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) and human dermal fibroblast (h-DFs) were embedded in fibrin hydrogel in a 3:1 ratio and cultured for 7 days within uHeart. The cardiac model developed a synchronous beating and the electrical activity was recorded through paired electrodes specifically inserted. We preliminary calibrated the system by assessing the pro-arrhythmic effect of known compounds (i.e. Verapamil, Sotalol and Terfenadine) on uHeart electrical activity. Aspirin and DMSO were used as negative control and vehicle, respectively. As expected, Terfenadine and Sotalol prolonged the repolarization time of cardiac microtissues at 100-1000 nM and 10-60 µM, respectively. In contrast, Verapamil decreased the FP duration and both Aspirin (up to 100 µM) and DMSO (up to 0.5% w/v) did not alter the beating properties. uKnee provides 3D cartilage-like constructs with either physiological (10%, 1Hz), or hyper-physiological compression (30%,1 Hz). The latter is able to elicit OA pathogenesis by mechanical factors. Primary human articular chondrocytes were embedded in a poly(ethylene-glycol)-based hydrogel and cultured in uKnee under static chondrogenic conditions for 2 weeks. Deposition of a cartilage-like matrix was demonstrated by immunofluorescence staining (i.e. Aggrecan, Collagen II) and a stable cartilage phenotype was evidenced by the increased expression of specific genes (i.e. ACAN, PRG4, ATX, FRZB and GREM1). After maturation, constructs were subjected to additional 7 days of confined compression at both intensity levels. Hyper-physiological compression induced OA-like traits and significantly enhanced catabolic and inflammatory response, as evidenced by MMP13 and IL8 gene expression. Responses to 4 drugs already used in the clinic (i.e. Rapamacyn, Celecoxib, IL-1Ra and dexamethasone) were assessed and resulted consistent with data from animal studies, probing the potential of uKnee as anti-OA drugs screening platform. Our new technology (uBeat) allows to develop beating OOC as powerful and reliable pre-clinical tools for efficient in vitro drug screening and disease modelling.
drug screening, organs on chip
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11311/1121280
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