三维类器官细胞培养系统

approaches have been applied to ensure proper medium supply, namely orbital shaking,11 spinning flasks6 and mini-bioreactors.10 However, applying fluidic systems (micro- or millifluidic) have not been considered, so far. In case of the hMOs, we hypothesize that keeping organoids under continuous orbital shaking as per published protocols5 might not be sufficient to ensure a proper supply of nutrients and oxygen. Therefore, to improve the quality of hMOs we investigated the effects of applying a continuous medium flow during culture. In this study we used the “Quasi Vivo” (QV, Kirkstall, UK) millifluidic system rather than a microfluidic device14 for a number of reasons. First, following the concept of allometry, the size of an organoid should range between approximately 0.5 to 2 mm in order to exhibit physiologically scaled metabolism (oxygen consumption).15,16 In addition, millifluidic systems such as the QV allow the application of relatively high flow rates ensuring proper nutrient and oxygen supply without exposing the organoid to a high shear force due to the flow itself. This is due to a well-like design in which the medium inlet and outlet are located in the chamber lid whereas the organoid is placed a variable distance from the medium inlet.17,18 Finally, given their high volume to surface ratio, millifluidic systems do not require frequent media changes, thus organoid manipulation is reduced to a minimum during culture. Besides various applications in 2D cultures, the QV millifluidic system has been successfully used to culture liver organoids13,19 and 3D cardiac constructs20 derived from human (adult) stem cells. In this study, we established a stable midbrain organoid culture under millifluidic conditions and compared it to the state-of-the-art procedure of continuous orbital shaking using both a computational fluid dynamics (CFD) and an experimental approach. The CFD analysis was performed to determine if differences in calculated oxygen profiles in the two experimental set-ups could be used to expla