Millifluidic culture improves human midbrain organoid vitality and differentiation

Abstract: Human midbrain-specific organoids (hMOs) serve as an experimental in vitro model for studying the pathogenesis of Parkinson’s disease (PD). In hMOs, neuroepithelial stem cells (NESCs) give rise to functional midbrain dopaminergic (mDA) neurons that are selectively degenerating during PD. A limitation of the hMO model, and of organoid models in general, is an under-supply of oxygen and nutrients to the densely packed core region, which leads to extensive cell death and eventually to a “dead core”. To reduce this phenomenon and to improve general culture conditions for the model, we applied a millifluidic culture system that ensures media supply by continuous laminar flow.

Since oxygen supply is a limiting factor in organoid cultures, we developed a computational model of oxygen transport and consumption in order to predict oxygen levels within the hMOs. The modelling predicts higher oxygen levels in the hMO core region under millifluidic conditions. In agreement with the computational model, a significantly smaller “dead core” was observed in hMOs cultured in a bioreactor system compared to those ones kept under conventional shaking conditions. Besides the reduced “dead core” size, the differentiation efficiency from NESCs to mDA neurons was elevated in hMOs exposed to medium flow. Increased differentiation involved a metabolic maturation process that was further developed in the millifluidic culture. Overall, bioreactor conditions improve hMO quality worth considering in the context of advanced PD modelling.