The artificial gut : integrating in vitro models of the human digestive tract with mass spectrometry
Currently, static in vitro cell culture assays are used in early phases of drug development, food research and hazard identification of chemicals. However, these in vitro models lack organ specific functionality, hampering mechanism-based research needed for novel drug development and next generation risk assessment. Recent advances in microchip- and bio-engineering enabled the development of organ-on-a-chip models. The main technical advantages of organ-on-a-chip technology are the ability to spatiotemporally control the microenvironment and the low reagent consumption. On the other hand, micro-engineered organ-on-a-chip models may still lack robustness for reproducible and bio-relevant studies. In addition, also the low flow rates present a major challenge of organ-on-a-chip technology: how to detect drug uptake and compound metabolism in real-time and at a high temporal resolution at the microscale.In this thesis a dynamic intestinal cell culture device is coupled to highly advanced mass spectrometry equipment, aiming for automated and online analysis of translocation of drugs, natural toxins and nanoparticles, while maintaining the bio-integrity of the cell system. Apart from technical robustness challenges, the fundamental chemical incompatibility between complex biological systems used in cell culturing and the clean sample requirements of sensitive advanced analytical instrumentation need to be resolved. Furthermore, the dynamic cell culture model is placed outside of an incubator for the integration with the mass spectrometer. However, physiological relevant temperatures and pH levels still need to be regulated for a reliable biological experiment. As well as the evaluation of the barrier integrity of the intestinal cell layer an aspect largely overlooked by current literature.In Chapter 2 an overview is provided of the current knowledge regarding analytical techniques integrated with organ-on-a-chip systems. In Chapter 3a a dynamic flow-through in vitro model was developed to predict permeability across the intestine using a well-known model drug, verapamil, to benchmark our results against literature. In Chapter 3b the stereoselective permeability of the natural mycotoxin ergotamine was examined using the model established in chapter 3a. The dynamic flow-through in vitro model system was expanded in Chapter 4. A novel bio-integrated online total analysis system for intestinal absorption and metabolism was developed by successfully interfacing a flow-through transwell with electrospray ionization mass spectrometry. The main advantages of this system being the integration of a relevant biological model with fully automated online analysis, which was missing before. Chapter 5 combines a digestion-on-a-chip with the bio-integrated analysis system developed in chapter 4, including the complexity of digestion creating a more complete bio-availability model. In Chapter 6 the integration of the flow-through transwell with inductively coupled plasma mass spectrometry was realized for the evaluation of the translocation of model gold nanoparticles. Lastly, in Chapter 7 a general discussion on the topics described in this thesis is provided and look to the future of organ-on-a-chip technology, with special emphasis on the integration with mass spectrometry analysis.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
| Published: |
Wageningen University
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| Subjects: | Life Science, |
| Online Access: | https://research.wur.nl/en/publications/the-artificial-gut-integrating-in-vitro-models-of-the-human-diges |
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| Summary: | Currently, static in vitro cell culture assays are used in early phases of drug development, food research and hazard identification of chemicals. However, these in vitro models lack organ specific functionality, hampering mechanism-based research needed for novel drug development and next generation risk assessment. Recent advances in microchip- and bio-engineering enabled the development of organ-on-a-chip models. The main technical advantages of organ-on-a-chip technology are the ability to spatiotemporally control the microenvironment and the low reagent consumption. On the other hand, micro-engineered organ-on-a-chip models may still lack robustness for reproducible and bio-relevant studies. In addition, also the low flow rates present a major challenge of organ-on-a-chip technology: how to detect drug uptake and compound metabolism in real-time and at a high temporal resolution at the microscale.In this thesis a dynamic intestinal cell culture device is coupled to highly advanced mass spectrometry equipment, aiming for automated and online analysis of translocation of drugs, natural toxins and nanoparticles, while maintaining the bio-integrity of the cell system. Apart from technical robustness challenges, the fundamental chemical incompatibility between complex biological systems used in cell culturing and the clean sample requirements of sensitive advanced analytical instrumentation need to be resolved. Furthermore, the dynamic cell culture model is placed outside of an incubator for the integration with the mass spectrometer. However, physiological relevant temperatures and pH levels still need to be regulated for a reliable biological experiment. As well as the evaluation of the barrier integrity of the intestinal cell layer an aspect largely overlooked by current literature.In Chapter 2 an overview is provided of the current knowledge regarding analytical techniques integrated with organ-on-a-chip systems. In Chapter 3a a dynamic flow-through in vitro model was developed to predict permeability across the intestine using a well-known model drug, verapamil, to benchmark our results against literature. In Chapter 3b the stereoselective permeability of the natural mycotoxin ergotamine was examined using the model established in chapter 3a. The dynamic flow-through in vitro model system was expanded in Chapter 4. A novel bio-integrated online total analysis system for intestinal absorption and metabolism was developed by successfully interfacing a flow-through transwell with electrospray ionization mass spectrometry. The main advantages of this system being the integration of a relevant biological model with fully automated online analysis, which was missing before. Chapter 5 combines a digestion-on-a-chip with the bio-integrated analysis system developed in chapter 4, including the complexity of digestion creating a more complete bio-availability model. In Chapter 6 the integration of the flow-through transwell with inductively coupled plasma mass spectrometry was realized for the evaluation of the translocation of model gold nanoparticles. Lastly, in Chapter 7 a general discussion on the topics described in this thesis is provided and look to the future of organ-on-a-chip technology, with special emphasis on the integration with mass spectrometry analysis. |
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