Multicellular spheroids (MCS) are considered as the most promising three dimensional (3D) in-vitro model which will narrow down the gap between in-vitro two-dimensional culture and in-vivo animal models. They exhibit physiologically relevant cell-cell and cell-matrix interactions, and present similar gene expression, heterogeneity and structural complexity as in-vivo tissues. Multicellular spheroids have been attempted for drug screening and evaluation, mechanical studies on cancer cell invasion and migration, and regeneration medicine. However, fabrication of uniform-sized MCSs at a high throughput platform, and evaluation of MCSs for clinical relevance are two main challenges. In this thesis, thermally responsive microgels were employed as physical supports to culture multicellular spheroids from both tumor cells and stem cells, which are potentially applied in anti-cancer drug evaluation, tissue engineering and regeneration medicine. The thermally reversible poly (N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-AA)) microgels were first employed to fabricate HeLa MCSs. This microgel approach restricted cell mobility at a lower initial cell density due to a large volume in the microgel networks, which resulted in uniform-sized spheroids formation compared to non-adhesive culture. Moreover, because of thermal reversibility of this microgel, spheroids were released from the physical supports via cooling down the system to room temperature. After demonstrating the formation of tumor spheroids in the microgel, HeLa cells were further encapsulated inside microgel-droplets generated from flow focusing microfluidics to obtain controllable uniform-sized spheroids. This approach combined the benefit of using thermal sensitive microgels as physical supports for MCS formation and droplet generation at a high throughput platform. Highly uniform-sized MCSs were obtained through this method. Importantly, the MCSs were easily released from the droplets by reducing the culture temperature to room temperature without using strong chemical or enzyme reagents. This approach may be used for generation of uniform-sized MCSs for drug screening and evaluation. The microenvironment generated from the microgel plays an important role in MCS formation. The key characteristics of the microenvironment, such as surface charge density, hydrophobicity, mechanical strength, and the microstructure of the microgels, were investigated by synthesizing a range of poly(N-isopropylacrylamide) (P(NIPAM)) based microgels, including P(NIPAM), P(NIPAM-co-methacrylic acid) (P(NIPAM-MAA)), P(NIPMAM-co-acrylic acid) (P(NIPAM-AA)), P(NIPAM-co-malic acid) (P(NIPAM-MA)) and P(NIPAM-co-itaconic acid)(P(NIPAM-IA)). It was found that the moderate negatively charged surface with high hydrophilicity P(NIPAM-IA) microgels was beneficial for cellular growth. The high or low charge density resulted in slow cell proliferation. The hydrophobicity of microgels had a negative impact on cell growth. The large pore size of the P(NIPAM-IA) networks also allowed cell migration which promoted MCSs formation. Different cell types (HEK 293, U87, HeLa and mesenchymal stem cells) have been demonstrated to successfully form MCSs within the P(NIPAM-IA) microgel. The thermal sensitive microgels were further applied to form stem cell MCSs. Human cardiac stem cells (hCSCs) were cultured in the P(NIPAM)) based microgel networks including P(NIPAM-co-dimethyl amino ethyl methacrylate) (P(NIPAM-DMAEMA)), P(NIPAM-IA), (P(NIPAM-co-2-hydroxyethyl methacrylate) (P(NIPAM-HEMA)), P(NIPAM-co-poly(ethylene glycol) methyl ether acrylate) (P(NIPAM-PEGA)). These microgels displayed different charges (cationic, anionic, and neutral) and different degrees of hydrophobicity. Through evaluation of hCSCs viability, proliferation and release of regenerative factors, P(NIPAM-IA) was identified as one of the best candidates for forming hCSCs spheroids because of its negatively charged surface with high hydrophilicity. The thermal reversibility of P(NIPAM-IA) renders it as injectable hydrogels. Initial results showed that injection of this microgel into mice did not elicit immune system responses, reduced myocardial apoptosis and promoted angiogenesis in the mice. In summary, we have fabricated MCSs in different types of thermal responsive microgels through either physical control of the uniform size by confining cells in the microgel-droplets generated from microfluidics or fine tune of the microenvironment for MCS formation. The P(NIPAM-IA) microgel with moderated anionic charge and high hydrophilicity was found to promote MCSs formation. This microgel did not elicit any immune response, which indicates the potential of using this microgel for future clinical studies.Thesis (Ph.D.) -- University of Adelaide, School of Chemical Engineering, 201
