Human iPS derived progenitors bioengineered into liver organoids using an inverted colloidal crystal poly (ethylene glycol) scaffold.

Generation of human organoids from induced pluripotent stem cells (iPSCs) offers exciting possibilities for developmental biology, disease modelling and cell therapy. Significant advances towards those goals have been hampered by dependence on animal derived matrices (e.g. Matrigel), immortalized cell lines and resultant structures that are difficult to control or scale. To address these challenges, we aimed to develop a fully defined liver organoid platform using inverted colloid crystal (ICC) whose 3-dimensional mechanical properties could be engineered to recapitulate the extracellular niche sensed by hepatic progenitors during human development. iPSC derived hepatic progenitors (IH) formed organoids most optimally in ICC scaffolds constructed with 140 µm diameter pores coated with type I collagen in a two-step process mimicking liver bud formation. The resultant organoids were closer to adult tissue, compared to 2D and 3D controls, with respect to morphology, gene expression, protein secretion, drug metabolism and viral infection and could integrate, vascularise and function following implantation into livers of immune-deficient mice. Preliminary interrogation of the underpinning mechanisms highlighted the importance of TGFß and hedgehog signalling pathways. The combination of functional relevance with tuneable mechanical properties leads us to propose this bioengineered platform to be ideally suited for a range of future mechanistic and clinical organoid related applications.

Diffusion-mediated in situ alginate encapsulation of cell spheroids using microscale concave well and nanoporous membrane.

Here, we present a novel and simple process of spheroid formation and in situ encapsulation of the formed spheroid without intervention. A hemispherical polydimethylsiloxane (PDMS) micromold was employed for the formation of uniform sized spheroids and two types of nano-porous membrane were used for the control of the crosslinking agent. We characterized the transport properties of the membrane, and the selection of alginate hydrogel as a function of gelation time, alginate concentration, and membrane type. Using the developed process and micromold, HepG2 cell spheroids were successfully formed and encapsulated in alginate without replating. This method allows spheroid encapsulation with minimal damage to the spheroid while maintaining high cell viability. We demonstrate the feasibility of this method in developing a bio-artificial liver (BAL) chip by evaluating viability and function of encapsulated HepG2 spheroids. This method may be applied to the encapsulation of several aggregating cell types, such as ß-cells for islet formation and stem cells for embryonic body preservation, or as a model for tumor cell growth and proliferation in a 3D hydrogel environment.