Three-dimensional recapitulation of neuroblastoma : from model to drug screening platform

Neuroblastoma is one of the most common yet fatal paediatric cancer. Nevertheless, few drug treatments are available in the market. One of the difficulties is the drug responses reported in 2-D in vitro tests does not always accurately reflect those observed in 3-D in vivo models. As the process consumes time, money and animal lives, a 3-D in vitro drug testing bioreactor recapitulating neuroblastoma tumours are needed to screen out any unsuccessful drug candidates before they enter in vivo tests. In this thesis, a 3-D in vitro model comprised neural stem cells (NSCs) or neurob- lastoma cells (SH-SY5Ys) and human umbilical vein endothelial cells (HUVECs) were initially optimised and developed in the laminin-rich hydrogel matrix to recapitulate neuroblastoma tumours. Next, a modified polydimethylsiloxane bioreactor (TissueFlex®) was developed to extend the viability of the 3-D in vitro model by enhancing the mass transport of nutrients and oxygen. The bioreactor was continuously perfused with a supplemented endothelial basal medium (EGM-2) co-treated with cisplatin, retinoic acid (RA), both cisplatin and RA, or EGM-2 as a control for three weeks. Cell morphology, cell viability and expressions of markers (TuJ1, Nestin and CD31) were measured weekly. Results were compared to parallel cultures of 2-D monolayer and 3DFlo® automatic cell culture systems. Our results show that hydrogel fragmentation allowed the formation of concentrated laminin and cellular networks along the gel granule boundaries with neurite extensions observed. Besides, cell surface marker expressions suggested an optimal NSC-HUVEC seeding density ratio of 3:1, cultured in 100% EGM-2 medium. The incorporation of perfusion culture to the model gave rise to significant increases in the number of cells and the expression of Nestin compared to static culture. After 3 weeks, only 2-D cultures showed a significant decrease in marker expressions upon treatment with cisplatin or cisplatin and RA in combination. Also, drug treatments were significantly less effective in 3-D bioreactors than in 2-D cultures. This suggests that the 3-D interactions between cells and hydrogel have created a microenvironment that recapitulates phenomena present in neuroblastoma and provides resistance to cancer therapeutics. Our 3-D in vitro model and perfusion bioreactor have implications for a more efficient drug discovery process by closing the gap between cell culture and physiological tissue investigations. These can be applied towards personalised medicine, where a patient's biopsy specimen can be tested in the system against a series of drugs to identify ones giving rise to optimal therapeutic outcomes.