Development of a compact parallel bioreactor combining mechanical and electrical stimulation for tissue engineering applications and mechanotransduction investigations

In tissue engineering (TE), physical stimuli, such as stretch or electrical pulses, have been demonstrated to be crucial for supporting several biological processes, e.g. for promoting the cell differentiation or the tissue maturation. Bioreactors, designed for providing in vitro physical stimuli, represent powerful tools to elucidate defined cellular mechanotransduction pathways. Indeed, they allow for investigating the relationship between in vivo-like physiological or pathological chemical and physical cues and cell response by enabling the setting of closely controlled and monitored 3D dynamic conditions, hence overcoming the limits of static cell cultures as well as ensuring standardization of the processes. In particular, bioreactors for providing mechanical stimulations are usually composed of a culture chamber, an actuation unit, and a control unit. The actuation unit, in combination with suitable clamping/support components, allows for the deformation of the cultured cells/constructs. Specifically, cyclic stretching can be useful for investigating the development and maturation of tissues such as tendons, ligaments, vessels, and cardiac muscle. A further physical stimulus that could be provided by inserting electrodes within the culture chamber is the electrical stimulation, which was shown influencing cell attachment, migration, proliferation, differentiation, and gene expression. The bioreactors actually available on the market can provide tunable mechanical or electrical stimulations to several samples in parallel. However, systems that combine mechanical and electrical stimuli are usually cumbersome and can house just few samples at once. In this context, the aim of this thesis was to design a compact bioreactor prototype for providing combinable mechanical and electrical stimulation to several constructs, with the final aim of supporting mechanotransduction investigations. The bioreactor was designed by following specific requirements, such as allowing to house simultaneously multiple independent constructs consisting of cell-loaded hydrogel, presenting low-volume culture chambers, being autoclavable, easy to assembly, simple to use, and suitable to be placed under the microscope. For the culture chamber, a customized lid, including mechanical and electrical parts, to be superimposed on a standard 12-well plate was designed (Solidworks). In detail, the system for providing mechanical stimulation is composed of a horizontal sliding part and a fixed counterpart where couples of vertical pillars are attached. Constructs, based on cells encapsulated in hydrogels, compact around the couples of pillars and, subsequently, can be exposed to stretch by moving the sliding part coupled with the actuation unit. Moreover, the lid presents holes for inserting within each well two electrodes, to be connected to an external electrical stimulator. The bioreactor was 3D printed and all design requirements have been checked and verified in terms of dimensional tolerances and assembly process flow. In the near future, once manufactured by using cytocompatible and autoclavable materials, the bioreactor reliability and performances will be tested for TE applications involving cell and tissue cultures subjected to different electromechanical stimulation protocols.