[Thesis]High-throughput processing and analysis of marine-based biomaterials and cells
As the field of Tissue Engineering (TE) progresses, new technology are essential to accelerate the identification of potentially translatable approaches for the repair of tissues damaged due to disease or trauma. The development of high-throughput and combinatorial technologies is helping to speed up research that is applicable to all aspects of the TE paradigm. In this thesis, interactions of cells with many diverse materials in both two- and three-dimensions was assessed rapidly through the use of superhydrophobic (SH) chips for rapid outcome measurements of cell-material or cell-cell combinations. The main hypothesis is that flat biomimetic SH surfaces patterned with transparent superhydrophilic (SL) spots were used to individual pattern biomaterials with precise shape and pre-determined height, by controlling the volume dispensed in each wettable spot. Initially, distinct combinations of nanostructured films were produced using layer-by-layer methodology and their morphological, physicochemical, and biological properties were analyzed using glass slides and then validated on-chip. Inspired by the composition of the adhesive proteins in mussels, thin films containing dopamine-modified hyaluronic acid were studied. The flat configuration of the SH chip allowed to perform a series of nondestructive and non-conventional measurements directly on the individual spots. In situ adhesion properties were directly measured in each wettable spot, showing that nanostructured films richer in dopamine promote the adhesion compared to control films (hyaluronic acid and alginate films – two polysaccharides often regarded as good natural adhesives – were assembled in parallel). In vitro tests showed an enhanced cell adhesion for the films with more catechol groups. Combining two biomimetic concepts we developed devices with multifunctional capabilities. One approach was based on two-sided film made almost entirely from polystyrene onto which the properties of both lotus leaves and mussel adhesive were incorporated. On one side of the film, imparting micro and nanometer scale hierarchical roughness yields superhydrophobicity and water repellency, providing rapid fluid flow and the basis for microfluidic devices, as such synthetic blood or fluid flow vessels. On second approach, SH microarray based on the so-called lotus effect were produced, onto which arrays of micro-indentations allow the fixing of liquid droplets, based on the rose-petal effect. Such platforms were able to sustain arrays of quasi-spherical microdroplets, allowing the isolation and confinement of different combinations of substances and living cells. Distinct compartmentalized physical, chemical, and biological processes were monitored individually in each droplet. In addition, taking the advantage of controlled positional adhesion and minimum contact with a solid substrate, we developed a novel hanging spherical drop system for anchoring arrays of droplets of cell suspension. By facing the chip downward it was possible to generate independent spheroids bodies in a high throughput manner, in order to mimic in vivo tumor models in a lab-on-chip scale. The system was validated for drug screening purposes and the toxicity of the anti-cancer drug doxorubicin in cell spheroids was tested and compared to monolayer cells culture. Finally, high-throughput fabrication of alginate hydrogel particles of specific sizes and shapes was developed using a droplet microarray. The method was based on the formation of arrays of droplets of pre-hydrogel solutions on SH-SL patterns using the process of discontinuous de-wetting, followed by their gelation via the parallel addition of the crosslinker to the individual droplets via the sandwiching method. The viability of living cells incorporated within the hydrogel particles was evaluated showing to be higher during the long-term cultivation than in the case of cells cultured in the bulk three-dimensional hydrogel matrix. In conclusion, SH platforms patterned with wettable spots used in this thesis proved to be compatible with a complete study of both two- and three-dimensional biomaterial-cell interactions, comprising a wide set of factors as biomaterials characterization and in vitro testing. Although much of the work performed is only applicable for in vitro studies, future methods may translate into rapid screening of these approaches in vivo.
Author: Neto, Ana Isabel Morais
Advisor: Mano, J. F.