Pappalardo, Francesco
(2022)
Gelatin methacrylate hydrogel microparticle-based 3D platform for elasticity-driven multi-lineage differentation of human mesenchymal stem cells.
PhD thesis, University of Nottingham.
Abstract
Understanding the mechanisms of the fate of stem cells is one of the major cornerstones of regenerative medicine research. Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into muscle, cartilage, bone, fat and nerve cells, and the complex mechanisms that they undergo are still not fully understood. Advances in cell signalling and developmental biology provided rudimentary pathways to differentiate MSCs in 2D culture to the aforementioned cell types, but the cellular microenvironment contains not just chemical signals but also those that are mechanical in nature. Previous research showed that matrix stiffness plays a significant role in MSC differentiation and that substrates can be tuned in their mechanical properties to tailor MSC lineage determination. However, most of the research performed in this area are in a 2D format, where it does not capture the 3D interaction effects of cells in culture and other physical aspects of the biomaterials such as curvature. Transitioning into a 3D platform can enable us to determine more accurately the physiological nature of stem cell behaviour.
In this thesis, a 3D culture platform has been produced using hydrogel microparticles made from methacrylated gelatin (GelMA). GelMA was selected as a material due to its biocompatibility, interactions with cellular integrins, and more importantly its tuneability of matrix stiffness. The latter property is essential to translate the 2D culture methods of varying matrix stiffness into a 3D format, where MSCs can be observed upon adhesion to these GelMA microparticles. Several ultra-porous microparticles of different sizes (170-760 μm) and modulus (0.23-130 kPa) were produced by tuning the dispersed and continuous phases of the co-flow microfluidic system used for their fabrication. An operability range of 5-13% w/v GelMA concentration in the dispersed phase, along with mineral oil in the continuous phase containing 1% w/v Span 80 as a surfactant was used. GelMA solution flow rates of 1-5 mL/h and 50-200 mL/h of mineral oil were also seen as viable process factors that produced acceptable, nearly monodispersed microparticles. Beyond these concentrations and flow rates, either highly heterogeneously sized microparticles were fabricated or the needle was blocked disabling the production of microparticles. Using these microparticles, MSCs were seeded and cultured for 7 days without any exogenous differentiation factors supplemented with the media. With only matrix stiffness therefore as a differentiation stimulus, the differentiation of MSCs could be tuned based on the surface modulus of the microparticle: 0.2-1.5 kPa for neurogenic, ~6 kPa for adipogenic, ~18 kPa for myogenic, and ~44 kPa for osteogenic lineages in preliminary studies. These data have been confirmed through immunocytochemical methods by staining for relevant markers of differentiation.
With this result, a successful platform could be used in performing 3D culture of MSCs in a stiffness-controlled manner where differentiation is directed through the matrix modulus alone. Such significant results could provide a new 3D biomaterial for MSC culture and differentiation, and these GelMA hydrogel microparticles could be utilised in suspension culture of MSCs with the aforementioned material as a carrier. This would have application in clinically relevant production of MSCs for regenerative medicine use.
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