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Digital archive of theses discussed at the University of Pisa


Thesis etd-02252010-172737

Thesis type
Tesi di dottorato di ricerca
Thesis title
SUITE an Innovative Bioreactor Platform for in vitro Experiments
Academic discipline
Course of study
tutor Prof.ssa Ahluwalia, Arti Devi
  • Bioreactor
  • cell culture
  • control
  • high throughput
  • pressure
  • sensor
  • stimuli
Graduation session start date
In-vitro cell cultures are a fundamental step in preclinical drug testing and are of great interest to the pharmaceutical industry. The most common method for culturing cells is in cell culture incubators. These are large and cumbersome and all mechanical stimuli are absent. They are nevertheless used ubiquitously and their results quoted as "standards" of in-vitro protocols. Several alternative culture methods have been proposed, and many systems are currently available commercially. Indeed, systems and devices for maintaining cells and tissues in controlled physical conditions, or bioreactors, have become an important tool in many areas of research. This is not only due to the growing interest in tissue engineering but also because it is now being increasingly recognised that cells respond not only to their biochemical, but also to their physical environment, and both cues are necessary to create a biomimetic habitat.
However most bioreactors for cell culture and tissue engineering are cumbersome and only provide a few cues such as flow or strain, allowing limited control and flexibility.

Since drug testing involves a large number of tests on identical cell cultures, a single well culture is inadequate and costly both in time and money. The High Throughput Screening (HTS), is a methodology for scientific experimentation widely used in drug discovery, based on a brute-force approach to collect a large amount of experimental data in less time and using less animals. The parallel nature of HTS makes it possible to collect a large amount of data from a small number of experiments and in a very short time. HTS, however, suffers from a significant problem that may affect the relevance of tests: the environment discrepancy problem. Another problem related with the actual drug testing and tissue engineering experiments is the enormous number of animals that have to be scarified every year.

The aim of this study was to develop a generic platform or SUITE (Supervising Unit for In-vitro TEsting) for cell, tissue and organ culture composed of two main components: a universal control unit and an array of bioreactor chambers. The platform provides a biomimetic habitat to cells and tissues since the environment in the chambers is controlled and regulated to provide biomechanical and biophysical stimuli similar to those found in-vivo. In this work I describe how a new concept of cell culture bioreactor was developed by integrating different technologies and research fields. The data extracted using this new cell culture approach is more predictive of the in vivo response with respect to the multi-well approach, particularly for drug related studies.

The starting point was a thorough analysis of currently used in-vitro methods; their pros and cons were assessed to exploit their advantages and overcome or circumvent their disadvantages.
As far as the culture chamber is concerned, the approach was to use the methods and materials commonly employed in microfluidic fabrication, but at scales compatible with classical culture systems such as petri-dishes and multiwells. This renders the bioreactors more amenable to use by biologists and enables the use of cell densities comparable with classic systems as well as the use of conventional assaying techniques. In most cases, the cell culture chambers are thus made out of PDMS (Polydimethylsiloxane), using soft-moulding with micro- or mini-machined masters, or what we call Soft Milli-molding. A system on a plate Multi Compartmental Modular Bioreactor (MCmB) was developed using this technology. The MCmB is a modular chamber for high throughput multi compartmental bioreactor experiments. It is designed to be used in a wide range of applications and with various cell types. A precise stimulus application is also very important to better understand the correlation between physical variables and pathologies allowing a more accurate study of the tissue physiology and pathologies. For this reason in these thesis three additional stimulation chambers for vascular and articular cartilage stimulation respectively were also designed and tested.
The control system was developed to be user-friendly, flexible and expandable to include new stimuli and was based on
modular components, including motors and sensors. Importantly a single software interface was designed to allow data acquisition and monitoring of several chambers in series or in parallel.

Using SUITE, high throughput experiments can be performed in an in vivo-like simulated environment for a long time to simulate different physiological or pathological scenarios or for toxicity testing of cells, tissues or in-vitro organ models.