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Development and characterisation of a functional 3D liver spheroid model
S176
Abstracts / Toxicology Letters 238S (2015) S56–S383
P07-007 Development and characterisation of a functional 3D liver spheroid model H. Gaskell 1,∗ , P. Sharma 1 , H. Colley 2 , C. Murdoch 2 , D. Antoine 1 , J. Sathish 1 , D. Williams 3 , S. Webb 1 1 University of Liverpool, Molecular and Clinical Pharmacology, Liverpool, United Kingdom 2 University of Sheffield, School of Clinical Dentistry, Sheffield, United Kingdom 3 AstraZeneca, Drug Safety, Cambridge, United Kingdom
In vitro liver models are a critical requirement for preclinical screening of drug candidates with hepatotoxic potential. In this regard, 3D liver models have recently emerged as an alternative to commonly used 2D techniques. 3D liver spheroids have been shown to have enhanced functional lifespan compared to 2D monocultures, as well as having potential to be used in high-throughput drug screening and chronic repeat-dose studies. However, the analyses of spatiotemporal function and internal structure of liver spheroids has received little attention. To address this concern we have developed and characterized the structure and function of a 3D liver spheroid model. Spheroids were formed from C3A hepatoma cells. Spheroids with a starting number of 750–2500 cells maintained a compact structure and steadily proliferated for at least 35 days in culture, highlighting potential for chronic repeat-dose studies. Histological analyses and transmission electron microscopy of sectioned spheroids revealed healthy cells with direct cell-cell contacts and tight junction formation. Additionally the spheroids had a compact in vivo-like internal structure and minimal necrosis up to day 33. MRP2 and Pgp were expressed on the canalicular membrane of cells in the spheroids after 4 days of culture and elongated bile canalicular structures formed by day 18 of culture. This indicates cell polarisation, advanced functionality and more in vivo-like cell morphology than standard 2D liver models. Compound penetration throughout the spheroid core was confirmed and dose-dependent toxicity occurred in response to model hepatotoxins. We have therefore successfully optimised a method for creating liver spheroids and validated initial structural and functional characteristics, providing an essential tool.
were combined by a microfluidic flow in a bioreactor the size of a microscopic slide. The 4-Organ-Chip consisted of two independent microfluidic circuits arranged on two levels, separated by a PET membrane. On the first, primary circuit, a primary human small intestinal model was inserted. The intestinal tissue was cultured in an integrated cell culture insert and provided a barrier function from the apical side of the intestine to the first circuit, allowing absorption. An on-chip micro-pump enabled the distribution from the basolateral side of the intestinal model to a liver equivalent, where potential substances could be metabolised. The microfluidic channel passed the bottom of the PET membrane, seeded with renal proximal tubule cells. This kidney model separated the first circuit from the second, excretory circuit. In this study, we combined these tissues with a skin biopsy, but this organ culture could also be replaced by any other organ equivalent, like neuronal tissue, lung tissue or others. The connected four organs were cultured for up to 28 days and showed a steady consumption of glucose and low LDH profiles during the complete culture period, providing evidence for a stable coexistence between the four tissues. The constitutive phase I and II enzymes were expressed in liver tissues and stayed constant over the time cultured. The intestinal tissues expressed glucose transporters and its barrier function was proven by expression of tight junction proteins and stable, near to physiologic TEER values. Renal proximal tubule cells showed polarisation, a steady expression of tight junctions and metabolic activity. Thus, a new tool for subsystemic substance testing with a potential for ADMET profiling has been developed. http://dx.doi.org/10.1016/j.toxlet.2015.08.512
P07-009 An in vitro evaluation of nicotine kinetics utilizing a human airway model (MucilairTM ) combined with an integrated multi-organ culture plate J. Ipema 1 , M. Gaca 1 , G. Phillips 1 , J. McKim Jr. 2,∗ 1 British American Tobacco, R&D Centre, Southampton, United Kingdom 2 IONTOX LLC, Kalamazoo, MI, United States
http://dx.doi.org/10.1016/j.toxlet.2015.08.511
P07-008 A microfluidic four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and kidney equivalents I. Maschmeyer 1,2,∗ , A. Lorenz 1 , A. Ramme 1 , T. Hasenberg 1 , K. Schimek 2 , J. Hübner 2 , R. Lauster 2 , U. Marx 1 1
TissUse GmbH, Berlin, Germany Technische Universität Berlin, Medical Biotechnology, Berlin, Germany 2
Ensuring good absorption, distribution, metabolism, excretion and toxicity (ADMET) properties is crucial to analyse if a drug reached its intended target and had a therapeutic effect without causing unacceptable toxicities. However, only few drug companies test ADMET characteristics in the early stages of drug discovery and development. Current in vitro models are lacking a systemic approach and therefore fail to predict the interaction of metabolites between organ cultures. Here, we present a new multi-organ approach to overcome this problem. Four human organ equivalents
The framework set out in the report entitled “Toxicity Testing in the 21st Century” (2007) focused on the need for the development of alternative methods to animal testing. Emphasis was placed on the use of human cells combined with adverse outcome pathways for determining adverse effects. Predicting systemic toxicity from in vitro data requires a platform that incorporates multiple organs interconnected via a fluidics network, in such a way that pharmacokinetic data can be integrated into the responses observed. The aim of this study was to evaluate a new Dynamic Multi-Organ Plate (DMOP) that provides the ability to use human tissues or cells, which are in communication via fluidics and dialysis. A simple transwell-based, two organ compartment model containing a human airway model (MucilAirTM ) was used to measure nicotine kinetics and biochemical effects. A standard 6-well plate was fitted with a fluidics/dialysis network. The dialysis membrane allows for exchange of test article and metabolites while maintaining each cell type under optimized media conditions. There is no net gain or loss of fluid. Nicotine (1000 M) was added to the apical side of the MucilAirTM model. Nicotine in the basolateral chamber diffused into the fluidics system via dialysis and was carried to the delivery well. A static sample from the basolateral and delivery chambers was collected every hour for 6 h. Perfusate from the fluidics system was collected at integrated one hour time inter-
Abstracts / Toxicology Letters 238S (2015) S56–S383
P07-007 Development and characterisation of a functional 3D liver spheroid model H. Gaskell 1,∗ , P. Sharma 1 , H. Colley 2 , C. Murdoch 2 , D. Antoine 1 , J. Sathish 1 , D. Williams 3 , S. Webb 1 1 University of Liverpool, Molecular and Clinical Pharmacology, Liverpool, United Kingdom 2 University of Sheffield, School of Clinical Dentistry, Sheffield, United Kingdom 3 AstraZeneca, Drug Safety, Cambridge, United Kingdom
In vitro liver models are a critical requirement for preclinical screening of drug candidates with hepatotoxic potential. In this regard, 3D liver models have recently emerged as an alternative to commonly used 2D techniques. 3D liver spheroids have been shown to have enhanced functional lifespan compared to 2D monocultures, as well as having potential to be used in high-throughput drug screening and chronic repeat-dose studies. However, the analyses of spatiotemporal function and internal structure of liver spheroids has received little attention. To address this concern we have developed and characterized the structure and function of a 3D liver spheroid model. Spheroids were formed from C3A hepatoma cells. Spheroids with a starting number of 750–2500 cells maintained a compact structure and steadily proliferated for at least 35 days in culture, highlighting potential for chronic repeat-dose studies. Histological analyses and transmission electron microscopy of sectioned spheroids revealed healthy cells with direct cell-cell contacts and tight junction formation. Additionally the spheroids had a compact in vivo-like internal structure and minimal necrosis up to day 33. MRP2 and Pgp were expressed on the canalicular membrane of cells in the spheroids after 4 days of culture and elongated bile canalicular structures formed by day 18 of culture. This indicates cell polarisation, advanced functionality and more in vivo-like cell morphology than standard 2D liver models. Compound penetration throughout the spheroid core was confirmed and dose-dependent toxicity occurred in response to model hepatotoxins. We have therefore successfully optimised a method for creating liver spheroids and validated initial structural and functional characteristics, providing an essential tool.
were combined by a microfluidic flow in a bioreactor the size of a microscopic slide. The 4-Organ-Chip consisted of two independent microfluidic circuits arranged on two levels, separated by a PET membrane. On the first, primary circuit, a primary human small intestinal model was inserted. The intestinal tissue was cultured in an integrated cell culture insert and provided a barrier function from the apical side of the intestine to the first circuit, allowing absorption. An on-chip micro-pump enabled the distribution from the basolateral side of the intestinal model to a liver equivalent, where potential substances could be metabolised. The microfluidic channel passed the bottom of the PET membrane, seeded with renal proximal tubule cells. This kidney model separated the first circuit from the second, excretory circuit. In this study, we combined these tissues with a skin biopsy, but this organ culture could also be replaced by any other organ equivalent, like neuronal tissue, lung tissue or others. The connected four organs were cultured for up to 28 days and showed a steady consumption of glucose and low LDH profiles during the complete culture period, providing evidence for a stable coexistence between the four tissues. The constitutive phase I and II enzymes were expressed in liver tissues and stayed constant over the time cultured. The intestinal tissues expressed glucose transporters and its barrier function was proven by expression of tight junction proteins and stable, near to physiologic TEER values. Renal proximal tubule cells showed polarisation, a steady expression of tight junctions and metabolic activity. Thus, a new tool for subsystemic substance testing with a potential for ADMET profiling has been developed. http://dx.doi.org/10.1016/j.toxlet.2015.08.512
P07-009 An in vitro evaluation of nicotine kinetics utilizing a human airway model (MucilairTM ) combined with an integrated multi-organ culture plate J. Ipema 1 , M. Gaca 1 , G. Phillips 1 , J. McKim Jr. 2,∗ 1 British American Tobacco, R&D Centre, Southampton, United Kingdom 2 IONTOX LLC, Kalamazoo, MI, United States
http://dx.doi.org/10.1016/j.toxlet.2015.08.511
P07-008 A microfluidic four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and kidney equivalents I. Maschmeyer 1,2,∗ , A. Lorenz 1 , A. Ramme 1 , T. Hasenberg 1 , K. Schimek 2 , J. Hübner 2 , R. Lauster 2 , U. Marx 1 1
TissUse GmbH, Berlin, Germany Technische Universität Berlin, Medical Biotechnology, Berlin, Germany 2
Ensuring good absorption, distribution, metabolism, excretion and toxicity (ADMET) properties is crucial to analyse if a drug reached its intended target and had a therapeutic effect without causing unacceptable toxicities. However, only few drug companies test ADMET characteristics in the early stages of drug discovery and development. Current in vitro models are lacking a systemic approach and therefore fail to predict the interaction of metabolites between organ cultures. Here, we present a new multi-organ approach to overcome this problem. Four human organ equivalents
The framework set out in the report entitled “Toxicity Testing in the 21st Century” (2007) focused on the need for the development of alternative methods to animal testing. Emphasis was placed on the use of human cells combined with adverse outcome pathways for determining adverse effects. Predicting systemic toxicity from in vitro data requires a platform that incorporates multiple organs interconnected via a fluidics network, in such a way that pharmacokinetic data can be integrated into the responses observed. The aim of this study was to evaluate a new Dynamic Multi-Organ Plate (DMOP) that provides the ability to use human tissues or cells, which are in communication via fluidics and dialysis. A simple transwell-based, two organ compartment model containing a human airway model (MucilAirTM ) was used to measure nicotine kinetics and biochemical effects. A standard 6-well plate was fitted with a fluidics/dialysis network. The dialysis membrane allows for exchange of test article and metabolites while maintaining each cell type under optimized media conditions. There is no net gain or loss of fluid. Nicotine (1000 M) was added to the apical side of the MucilAirTM model. Nicotine in the basolateral chamber diffused into the fluidics system via dialysis and was carried to the delivery well. A static sample from the basolateral and delivery chambers was collected every hour for 6 h. Perfusate from the fluidics system was collected at integrated one hour time inter-