The Laminar Flow Bioreactor was designed to reproduce laminar flow over cells in the

CONCLUSIONS

This PhD thesis was based on study of new cell-based technologies for medicine, tissue engineering and pharmacological research. In particular, our aim was to develop and test different biotechnological devices called bioreactors, designed to recreate, in vitro, the human physiological environment in terms of chemical and physical stimuli which are impossible to reproduce in classic cell culture conditions. These devices have the potential to provide information on local cell behavior and function and their development could lead to a multitude of applications from drug testing and development, tissue engineering, and basic research to the identification of new and alternative therapies for many disorders. The bioreactors were designed at the Interdepartmental Research Center “E. Piaggio” of Faculty of Engineering of Pisa University under the MCB Team leaded by Prof. Arti Devi Ahluwalia and tested in the Laboratory of Biomimetic Materials and Biological Tissues Engineering of Dr. Claudio Domenici at CNR Institute of Clinical Physiology in Pisa; the biological and pharmacological aspects were under the supervision and support of Dr. Vincenzo Calderone of Department of Psychiatry, Neurobiology, Pharmacology and Biochemistry of Faculty of Pharmacy at Pisa University. In these three years, my work was based in the testing and the development of three bioreactors: the Laminar Flow, the Array and the Multi-Compartmental Bioreactor. Each one has the possibility to reproduce an aspect of human physiology: laminar flow and shear stress present in vascular with the Laminar Flow Bioreactor; concentration gradients of numerous endogenous substances with the Array Bioreactor; physiological and biological complexity and interaction of different cell types in the Multi-Compartmental Bioreactor.

The Laminar Flow Bioreactor was designed to reproduce laminar flow over cells in the

chamber and analyze specifically the effect of this particular force without interference

of pressure, which is not possible in vivo or in traditional in vitro culture systems. The

application field is for vascular studies with the aim of a better comprehension of

Conclusions

194

physiological and patho-physiological mechanisms of the endothelium. We have investigated, in these experiments, production trends by Human Umbilical Vein Endothelial Cells (HUVECs) of the two most important endogenous mediators, Nitric Oxide (NO) and Endothelin-1 (ET-1), in response to different flow rates at different times obtaining a baseline for further studies. The experimentation described above was divided into two phases: one, in static conditions, where different compounds (acetylcholine, L-NAME, resveratrol, NS 1619, Iberiotoxin) we tested to have a base- line behaviour of drugs used in the presence or absence of activator/inhibitor of receptors/channels and understand the biological pathway/intracellular messengers involved in their mechanism of action; a second phase was directed towards analysis of the effect of flow and, in particular, of shear stress directly in endothelial cell culture with the Laminar Flow Bioreactor. With the results obtained from dynamic cell cultures, we can assert that the bioreactor can recreate laminar flow conditions similar to the physiological environment of the vascular system. Future studies can include the integration of mechanical stress with pharmacological stimulus to understand the cell behaviour and response, under chemical treatment, in a more physiological environment.

The Array Bioreactor presents particular characteristics to develop a concentration

gradient of one or more substance over cells thanks to its particular channel geometry

and flow in the culture chamber. The chamber was used to test the apoptotic effects

of two different concentrations of Hydrogen Peroxide (H

O

) on HUVECs with

fluorescence imaging techniques. In a preliminary phase, we tested the apoptotic

activity of a gradient of H

O

on HUVECs in culture. A series of tests were conducted in

static conditions to define times (tests performed for 30, 60 and 90 minutes) and

concentrations (0,1, 0,5 and 1,0 mM) of H2O2 to be used, then, in the Array

Bioreactor. Once results in static culture were obtained, the same treatment, with 0,5

and 1,0 mM of H

O

for 30 minutes, was performed in the Array Bioreactor. Here, a

luminous gradient directly proportional to hydrogen peroxide concentrations was

observed in cell culture chamber. These results provided a first validation of our

Conclusions

195

system. Our aim is to furnish a tool useful for comprehension of cell dynamics in response to concentration gradients in pharmacological and, particularly, in pharmacogenomic applications. In fact, the possibility to test different concentrations of the same compound or different compounds, at defined concentrations, at the same time can help to correlate the influence of genetic variations on drug response in patients by correlating gene expression or single-nucleotide polymorphisms with a drug's efficacy or toxicity.

Finally the last part of the thesis describes the Multi-Compartmental Bioreactor (MCB) which can be used to create a model of an organ or organism by incorporating a variety of cell types into interconnected compartments. It was designed to lodge four different cell types (pancreatic, hepatic, endothelial and adipose cells) with the use of particular rules called “allometric laws” which correlate different parameters (distribution volumes, organ dimensions, retention time). The final aim of this bioreactor is to approximate the organism that it has been designed to mimic and therefore offer the potential to replace or reduce the use of animals for the evaluation of disease mechanisms and effects of new drugs or chemicals, through the use of an

‘artificial organ or organism’. After a preliminary test phase to analyze flow distribution

and cell survival in the MCB, two different experiments were performed, one in

Leipzig, at the Laboratory of Cell Techniques and Applied Stem Cell Biology of BIOCITY

in Leipzig under the leadership of Prof. Augustinus Bader, and a second in Pisa, in our

laboratory at Institute of Clinical Physiology at CNR. In the first case, we used primary

murine hepatocytes and HUVECs: we have evaluated different metabolites such as

albumin and urea, useful to understand cell behavior. After initial studies in static

conditions, we performed tests in the MCB. Results obtained showed, fundamentally,

an increase of production of urea and albumin when the two cell types were in

connected cultures. The same studies were repeated in Pisa using human

hepatocarcinoma cell line, called HepG2, and HUVECs. Also in these experiments the

increase of albumin and urea concentrations was evident, confirming the same cell

behavior observed in Leipzig and providing us a validation of proof of concept of MCB.

Conclusions

196

This work paves the way towards (adipocytes and pancreatic islets) a better

comprehension of possible cell interactions and a definition of patho-physiological

disease models to be reproduced in our system. Our aim is to furnish a device where it

is possible analyze the cell complexity and the reciprocal behavior of various cell

typologies and test new compounds for a prediction of metabolite production and

their effects on different “cell targets”.