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I NDEX
Abstract
1General introduction
3Chapter I: Metabolism and metabolic disorders
1.1 Introduction 5
1.2 The organism energetic balance 5
1.3 Insulin 13
1.4 Pathologies connected to the glucose metabolism 19
1.5 Diabetes 19
1.6 Dysmetabolic syndrome X 31
1.7 The liver 33
1.8 Metabolism of glucose and fatty acids by the liver 36
1.9 General properties of postprandial (absorptive) state 36
1.10 The postabsorptive state 37
1.11 The fasted state 38
1.12 Physiology and regulation of fatty acid metabolism 40
1.13 Storage and release of fatty acids in adipocytes 41
1.14 Hepatic metabolism of fatty acids 41
1.15 Adipose tissue is an active endocrine organ 42
1.16 Conclusion 42
Bibliography 43
Chapter II: Tissue Engineering: application in Liver Engineering
2.1 Introduction 48
2.2 Tissue engineering 48
2.3 Cells 49
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2.4 Tissue culture 49
2.5 Engineering materials 50
2.6 Synthesis of tissue engineering scaffolds 51
2.7 Pressure Assisted Micro-syringe (PAM) 52
2.8 Bioreactors 56
2.9 Liver tissue engineering 57
2.10 Conclusion 60
Bibliography 61
Chapter III : Bioreactors for metabolic studies
3.1 Introduction 64
3.2 Systems for metabolic studies 64
3.3 New concept systems 67
3.3.1 Micro Cell Culture Analog (µCCA) 67
3.3.2 Multi-Compartmental Bioreactor (MCB) 71
3.4 MCmB: Assessment of modular bioreactor (MCmB) performance with respect to the fixed topology MCB system
76
3.5 Conclusion 80
Bibliography 82
Chapter IV: Materials and Methods
4.1 Introduction 84
4.2 Structure polymers 84
4.3 Structure geometry 87
4.4 Sterilization and functionalization 88
4.5 Cell types 89
4.6 First experimental protocol: Development of a model of hepatic culture that allows creation of an in vitro engineered three dimensional (3D) liver-like micro-environment optimized for use in the bioreactor.
90
4.7 Second experimental protocol: A) Identification of a composite medium to use for all cellular types in the MCmB and B) development of
91
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initial mono-compartmental models using the MCmB (dynamic conditions).
4.8 Third experimental protocol: Development of a three-compartmental connected-culture model (in dynamic condition) and study in comparison with the same connected-culture without hepatocytes.
93
4.9 Fourth experimental protocol: Study of the influence of increased glucose level on metabolic markers in the MCmB in order to simulate human hyperglycemia.
94
Bibliography 96
Chapter V: Results and Discussion
5.1 Introduction 97
5.2 First experimental results: Development of a model of hepatic culture that allows creation of an in vitro engineered three dimensional (3D) liver-like micro-environment optimized for use in the bioreactor
97
5.3 Second experimental results: A) Identification of a composite medium to use for all cell types in the MCmB and B) development of initial mono-compartmental models using the MCmB (dynamic conditions).
100
5.4 Third experimental protocol results: Development of a three- compartmental connected-culture model (in dynamic condition) and study in comparison with the same connected-culture without hepatocytes.
104
5.5 Forth experimental protocol results and discussion: Study of the influence of increased glucose level on metabolic markers in the MCmB in order to simulate human hyperglycemia.
108
General conclusion
page114
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1
Abstract
Cellular function in the native environment is influenced by a variety of biochemical factors, which interact to create complex signalling mechanisms.
This complex set of biochemical and mechanical signals that regulate cellular metabolic function in vivo is incompletely understood. A systematic examination of how changes in environmental conditions may lead to phenotype shifts will improve the understanding of cellular biology and pathology as well as be of benefit in developing appropriately targeted therapies. Recreating this level of control in vitro is challenging, but it requires generating and and characterising models that simulate in vivo circuitry.
The aim of this work was therefore to develop and optimize a compartmentalized modular bioreactor to faithfully represent salient aspects of internal organs and tissue metabolism, transport and kinetics. With the increasing recognition of the metabolic syndrome as being one the major health issue in the developed world, this work furnishes a basic validation of an adaptive and evolving tool able to resolve and understand aspects related to this epidemic disease and associated pathologies. The multi-compartimental modular bioreactor (MCmB) system is designed as an in vitro model of tissue metabolism so as to provide a means to interpret and cure metabolic disorders.
It could also be used as a test bed for different hypotheses explaining the triggering mechanisms behind diabetes. No previous study has examinated a multi-compartment model where cultures of insulin-secreting cells, hepatocites, adipocytes and endothelial cells are connected in a closed circuitrydy vascular type spaces. Only one example of a similar system has been reported, for application in pharmacokinetic modelling in liver.
The optimization of the MCmB was divided in several phases:
• Selection of the different cell types that can be used inside the MCmB and that present the best characteristics to mimic the conditions investigated and pathways of human metabolism
• Development of a model of hepatic culture that allows creation of an in vitro engineered three dimensional liver-like micro-environment optimized for use in the bioreactor (In collaboration with Drs Brunetto M., PhD; Gastroenterology- Hepatology research laboratories, S. Chiara Hospital, Pisa)
• Determination of optimum cell numbers for each selected cell type in order to reproduce the same condition and ratios between the different cell types present inside the human body, and scaling these quantities using allometric lows
• Identification of a composite medium to use for all cell types in the MCmB
• Development of an initial mono-compartmental model inside the bioreactor (dynamic conditions) for every cell type
• Development of a three-compartmental connected-culture model (in dynamic conditions) and study in comparison with the same connected- culture lacking in liver tissue
2 the MCmB in order to simulate human hyperglycemia (In collaboration with Prof Avogaro A., PhD; Clinic and Experimental Medicine research laboratories, University of Padova).
Considering that the liver is the most important organ that develops metabolic activity on endogenous and exogenous substances our first goal was developing a model of hepatic culture that allows the creation of an in vitro liver-like micro-environment optimized for use in the bioreactor. The PAM (Pressure Activated Microsyringe, system developed at the Interdepartmental Research Centre “E.Piaggio”) was the technique used for the microfabrication of three dimensional polymeric scaffolds (3D microscale structures) in order to give a topological input to the cells in culture and to increase the cell number per area (cell density). The polymeric scaffolds were realised with poly-lactide (PLLA) and Poly (lactide-co-glycolide) (PLGA), two biocompatible and biodegradable polyesters. We studied cell survival, proliferation and metabolism on these polymeric microscale structures with a well-defined geometry in comparison with films of the same polymers in static conditions. We performed experiments on 3D scaffolds in dynamic conditions too and compared the results with the static ones.
The results in static conditions on the 3D scaffolds demonstrated that microfabrication techniques allow an increase of the cell number per unit area (cellular density) and consequently increase of the surface-area-to volume ratio of the cellular mass once inserted in the bioreactor. The results on 3D hepatic tissue in dynamic culture were the first demonstration of enhanced cellular function status in the MCmB device.
In the second phase, after having optimized a three-dimensional model of the liver, we developed a three-compartmental dynamic model using liver, adipose and vascular tissue.
The main advantage of the MCmB is the possibility of simulating and inducing cell cross-talk between different tissues. We therefore tested the system using different levels of glucose and insulin concentration to simulate basal and postprandial conditions. The tissue response in the MCmB was similar to that observed in humans in-vivo.
The MCmB development studies showed firstly the presence of cell cross-talk in this new device and secondly the importance of considering cell cross-talk during in vitro investigations on cell function and response in complex diseases. Overall this bioreactor system is shown to be capable of approximating the in–vivo environment much better than classic static or dynamic monocultures.
