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Digital archive of theses discussed at the University of Pisa


Thesis etd-05102018-150001

Thesis type
Tesi di dottorato di ricerca
Thesis title
3D Microstructures for Liver-on-Chip and Gene Delivery Applications
Academic discipline
Course of study
tutor Prof. Barillaro, Giuseppe
  • 3D microstructures
  • gene delivery
  • liver-on-chip
  • replica molding
  • silicon electrochemical micromachining
Graduation session start date
Release date
Nowadays, considerable advances have been made in the development of bioengineered three-dimensional platforms that seek to faithfully replicate the complexity and functionality of human physiology and pathology in research laboratories. The high versatility of advanced micro and nanofabrication technologies, in term of implementation of different chemical, topographical, mechanical and microfluidic aspects, has been exploited for the fabrication of three-dimensional platforms suitable for a pletora of different applications, such as rigenerative medicine, tissue engineering, organ-on-chip for drug screening, gene terapy, and immunology.
In this thesis, two different microfluidic bioinspired platforms, one silicon-based and one polymer-based, have been successfully fabricated for liver-on-chip application. A microfabrication technology, namely silicon electrochemical etching, was successfully used for the fabrication of the silicon-based three-dimensional platform. More in details, a microfluidic device integrating electrochemically etched silicon micro-trenches
(width 4 µm, pitch 8 µm and depth 10 and 20 µm) was developed to reassemble the liver sinusoid microarchitecture thus allowing a cord-like arrangement of the mouse primary hepatocyte and, in turn, their long-term culturing and functional maintenance (i.e. 28 days) thank to the cell-cell interaction and the continuous culture medium flow that provide convective transport of nutrient and waste products. A drug commonly used to treat pain and fever, namely acetaminophen, was successfully used as drug model to demonstrate the effectiveness of the microfluidic device integrating the in-silicon micro-trenches for in-vitro hepatotoxicity testing in order to predict in-vivo hepatotoxicity. Moreover, silicon micro-trenches for human hepatocyte cell culture, featuring different pitches (i.e. 18, 28 and 58 µm) and depths (i.e. 30 and 85 µm), were designed and fabricated by electrochemical micromachining technology. Preliminary viability and morphological assessments of immortalized human hepatocyte cells, namely Hep G2, cultured on 30-µm-depth silicon micro-trenches for 7 days were carried out for evaluation of the more suitable micro-trench pitch for immortalized hepatic cells. Additionally, a polymer-based replication technology, namely replica molding,
was well exploited to create microfluidic bioinspired platforms for liver-on-chip application. In particular, the cell culture chamber of the microfluidic platform was designed on the basis of the architecture of the liver smallest functional unit, namely liver lobule, thus mimicking the lobule hexagonal shape, the radial sinusoid/hepatic-cord arrangement, and the physiological radial blood flow from the portal triads to the central vein. PDMS bioinspired microfluidic platforms (and theirs lids), featuring two different hexagonal-shape chamber areas (i.e. 1:5 and 23 mm^2) and two different line widths (i.e. 20 and 30 µm), were successfully fabricated by a two-step replica moulding process, using durable and reusable epoxy-resin masters replicating geometrical features of SU-8 masters fabricated by standard UV photolithography, for which all parameters were optimized in order to obtain 45 µm-high structures on silicon substrate, good adhesion
to the substrate as well as high sidewalls verticality. Preliminary tests for cell seeding density assessment were carried out by using cryopreserved human hepatocytes with three different seeding cell density (1.0 x 10^5 cell/mL, 2.0 x 10^5 cell/mL and 4.0 x 10^5 cell/mL) after embedding the collagen-treated PDMS microfluidic platform in a customized microfluidic setup.
Furthermore, silicon electrochemical etching in HF-based electrolyte solution was used for the fabrication of silicon-based three-dimensional platforms for immunological gene delivery. More in details, ordered arrays of silicon pillars, featuring either diameter about 1 µm, pitch 4 µm, depth between 5 and 30 µm, and apical surface rounded/sharp, or diameter about 1 µm, pitch 10 µm, depth between 5 and 15 µm, and rounded apical surface, were successfully fabricated by one-electrochemical-etchingstep, and both morphologically and optically characterized by using a scanning electron microscope and a reflectance optical line at normal incidence, respectively.
Finally, advances in silicon microstructuring technology by electrochemical etching have been also demonstrated in order to further improve the already high versatility of this method for the fabrication on 3D microstructures for cell-related applications. A silicon electrochemical etching able to set a novel record among either commercial or state-of-the-art silicon etching technologies in term of etching rate versus aspect
ratio was reported. More in details, the controlled electrochemical etching of high aspect-ratio structures (from 5 to 100) in silicon at the highest etching rates (from 3 to 10 µm/min) at room temperature adding an oxidant, namely H2O2, to a standard aqueous hydrofluoric acid electrolyte was successfully demonstrated. The presence of H2O2 dramatically changes the stoichiometry of the silicon dissolution process under anodic biasing without loss of etching control accuracy at the higher depths (up to 200 µm). It was shown that the presence of H2O2 reduces the valence of the dissolution process to 1, thus rendering the electrochemical etching more effective, and catalyses the etching rate by opening a more efficient path for silicon dissolution with respect to the well-known Gerischer mechanism, thus increasing the etching speed at
both shorter and higher depths. Furthermore, for the first time, we reported successful application of electrochemical etching technology for the controlled and simultaneous realization of ordered arrays of out-of-plane macropores, with diameter tuneable in the range 0.47-0.85 µm, interconected by high-density secondary in-plane pores, with diameter of about 250 nm, by electrochemical dissolution of n-type silicon in HF-based electrolyte with the presence of H2O2 through the synergistic use of back-side illumination, avalanche breakdown anodization voltage, and high-oxidizing-power chemical.