Chiara Canali

Fabrication of scalable and structured tissue engineering scaffolds using water dissolvable sacrificial 3D printed moulds.

One of the major challenges in producing large scale engineered tissue is the lack of ability to create large highly perfused scaffolds in which cells can grow at a high cell density and viability. Here, we explore 3D printed polyvinyl alcohol (PVA) as a sacrificial mould in a polymer casting process. The PVA mould network defines the channels and is dissolved after curing the polymer casted around it. The printing parameters determined the PVA filament density in the sacrificial structure and this density resulted in different stiffness of the corresponding elastomer replica. It was possible to achieve 80% porosity corresponding to about 150 cm(2)/cm(3) surface to volume ratio. The process is easily scalable as demonstrated by fabricating a 75 cm(3) scaffold with about 16,000 interconnected channels (about 1m(2) surface area) and with a channel to channel distance of only 78 μm. To our knowledge this is the largest scaffold ever to be produced with such small feature sizes and with so many structured channels. The fabricated scaffolds were applied for in-vitro culturing of hepatocytes over a 12-day culture period. Smaller scaffolds (6×4 mm) were tested for cell culturing and could support homogeneous cell growth throughout the scaffold. Presumably, the diffusion of oxygen and nutrient throughout the channel network is rapid enough to support cell growth. In conclusion, the described process is scalable, compatible with cell culture, rapid, and inexpensive.

Electrical impedance tomography methods for miniaturised 3D systems

Abstract In this study, we explore the potential of electrical impedance tomography (EIT) for miniaturised 3D samples to provide a non-invasive approach for future applications in tissue engineering and 3D cell culturing. We evaluated two different electrode configurations using an array of nine circular chambers (Ø 10 mm), each having eight gold plated needle electrodes vertically integrated along the chamber perimeter. As first method, the adjacent electrode configuration was tested solving the computationally simple back-projection algorithm using Comsol Multiphysics in time-difference EIT (t-EIT). Subsequently, a more elaborate method based on the “polar-offset” configuration (having an additional electrode at the centre of the chamber) was evaluated using linear t-EIT and linear weighted frequency-difference EIT (f-EIT). Image reconstruction was done using a customised algorithm that has been previously validated for EIT imaging of neural activity. All the finite element simulations and impedance measurements on test objects leading to image reconstruction utilised an electrolyte having an ionic strength close to physiological solutions. The chosen number of electrodes and consequently number of electrode configurations aimed at maximising the quality of image reconstruction while minimising the number of required measurements. This is significant when designing a technique suitable for tissue engineering applications where time-based monitoring of cellular behaviour in 3D scaffolds is of interest. The performed tests indicated that the method based on the adjacent configuration in combination with the back-projection algorithm was only able to provide image reconstruction when using a test object having a higher conductivity than the background electrolyte. Due to limitations in the mesh quality, the reconstructed image had significant irregularities and the position was slightly shifted toward the perimeter of the chamber. On the other hand, the method based on the polar-offset configuration combined with the customised algorithm proved to be suitable for image reconstruction when using non-conductive and cell-based test objects (down to 1% of the measurement chamber volume), indicating its suitability for future tissue engineering applications with polymeric scaffolds.

Impedance-based monitoring for tissue engineering applications

Impedance is a promising technique for sensing the overall process of tissue engineering. Different electrode configurations can be used to characterize the scaffold that supports cell organization in terms of hydrogel polymerization and degree of porosity, monitoring cell loading, cell proliferation as well as the spatial distribution of cell aggregates in 3D. We have previously shown that impedance measurements allow accurate determination of conductivity in physiological solutions independent of validation and analysis of a specific equivalent circuit. Similarly to a physiological solution, cell culture medium conductivity, and hence the measured impedance, can respond to proliferating or dying cells populating the scaffold. Impedance may therefore be a key parameter for monitoring the biochemical dynamics that modulate 3D mammalian cell cultures over time. Furthermore, the conductivity of the medium filling the pores of the scaffold can serve as the basis for porosity determination using Archie’s law. Different networks of structured or random channels and degree of porosity can be detected. In addition, by combining a number of two-, three- and four-terminal (2T, 3T, 4T) configurations, it is possible to obtain complementary information on spatial distribution of cells in a 3D scaffold. 2T- and 3T configurations also reflect the impedance at the interface between an electrode and cell-loaded scaffold (polarization impedance, Zp), which may convey a further degree of information about the biochemical phenomena taking place in that sub-volume.