• 3D cell culture
• cell culture
• ovarian cancer
• scaffold

The discovery and development of new drugs is a very lengthy and costly process that ranges between $800 million and $2 billion and can take up to 15 years, where almost a third of the promising compounds fail during phase II and phase III clinical stages, due to poor efficacy and safety issues. One of the main reasons for drug failure is inappropriate preclinical testing methods and in vitro models, which do not sufficiently produce information for prediction of drug efficacy and toxicity. Cell culture is an important and necessary, if not the most influent, process in drug discovery and cancer research but, since most research studies are still carried on two-dimensional (2D) cultures, the disclosure of new effective molecules and their approval is arduous. Novel and improved methods that implement this process are therefore indispensably needful. Three-dimensional (3D) cell culturing techniques suggest compelling evidence that much more advanced experiments can be performed yielding valuable and more reliable insights. This cell culture system allows reproducing more faithfully the cell environment mimicking that of a cell in vivo and providing more accurate data about cell-to-cell interactions, tumour characteristics, drug discovery and other types of diseases. To date many three-dimensional approaches exist, each providing their own advantages and applications. Scaffold based techniques, such as hydrogel-based support, are some of the 3D culture practice employed and present an array of benefits. 3D hydrogel scaffolds are unique because of their ability to mimic the extracellular matrix (ECM) while allowing soluble factors such as cytokines and growth factors to travel through the tissue-like gel.
The present research activity has been performed under a multidisciplinary approach that combines polymer chemistry, additive manufacturing and biology with the purpose of developing a 3D cell culture model for ovarian cancer that will allow, with further studies, to enhance and obtain more reliable results in drug discovery, toxicity evaluations and many other fields of medicine and chemistry. Two natural polymers, namely Chitosan and Alginate, were chosen for their proven biocompatibility, opposite charge at physiological pH and ready availability from renewable and sustainable sources, as matrices for the development of 3D Microstructured Polyelectrolyte Complex (mPECs) Hydrogels. The samples were fabricated employing additive manufacturing techniques that compared to conventional techniques, allow for a fine control of the external geometry and macro and microporosity of the polymeric structure, leading to the obtainment of customizable shapes and above all reproducible samples. Chitosan and Alginate were used in a mixture, to create hydrogels for the manufacture of microstructured scaffolds by means of Computer-Aided Wet-Spinning (CAWS). The CAWS technique is an additive manufacturing technique which, starting from polymer solutions or suspensions, allows to obtain structures with a multiscale porosity characterized by a macro-porous network, determined by the path of deposition of the polymeric fibre, and by a micro or nano- porosity, determined by the phase separation process induced by non-solvent which is at the basis of the solidification of the material. With this technique, scaffolds were manufactured with different compositions of the polymer blend of the two natural polysaccharides and the manufacturing parameters were optimized for each composition. 3D microstructure hydrogel based only on Chitosan ionically crosslinked with tripolyphophate (TPP) was also prepared by CAWS, in order to compare the influence of hydrogel composition on the physical-chemical, mechanical and biological features of the systems.
The swelling degree of the manufactured scaffolds was evaluated in physiological conditions, and the results suggested the presence of a diffuse and interconnected porous structure that further studies, to be preformed by scanning electron microscopy, will have to confirm. Furthermore, all fabricated the scaffolds preserved their physical integrity after a long period of incubation up to 21 days, a feature that can possibly make them suitable for the employment for long-term cell culture applications.
The biological evaluation of the developed 3D Hydrogels was started by analysing the cell adhesion response of the human ovarian cancer cell line A2780. The obtained results showed a significantly higher adhesion of cell onto the mPECs containing Chitosan and Alginate with respect to the hydrogels prepared with Chitosan only, thus suggesting an important role of the chemical composition in guiding cell response. Ongoing studies are devoted to assess cell proliferation and to investigate the influence of chemical composition on the physical-chemical and mechanical properties of the samples and to correlate such feature to cell response.