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 is an under-supply of oxygen and nutrients to the densely packed core region, which leads eventually to a "dead core". To reduce this phenomenon, we applied a millifluidic culture system that ensures media supply by continuous laminar flow. 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. Comparing the necrotic core regions in the organoids with those obtained from the model allowed an estimation of the critical oxygen concentration necessary for ensuring cell vitality. 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 that improve hMO quality are worth considering in the context of advanced PD modelling.

Allometric Scaling of physiologically-relevant organoids

“…As they are hollow, they are unlikely to suffer from necrotic cores 37 . On the other hand, non-luminal organoids such as brain and liver often possess non-viable centres, principally due to the lack of oxygen which is widely considered as the limiting metabolic resource for living cells 17,22 . Local oxygen levels also influence cell fate [38][39][40][41][42] , likely compromising tissue function and structural arrangement.…”

“…Referring to published data on brain organoids derived from human induced pluripotent stem cells (hiPSCs) 13,17 , we modelled both stem cell proliferation and organoid volume expansion to identify a range of experimental organoid masses and corresponding cell densities. In particular, cell proliferation was modelled using the Sherley model 23 :…”

“…Number of cells, average organoid radii, mass and cell densities at each time point investigated. Data on hiPSC generated brain organoids from Monzel et al 13 and Berger et al 17 .…”

“…10 12 cells/m 3 ) is about two orders of magnitude lower than those typically reported for such organoids (around 10 14 cells/m 3 ) which are close to physiological cell densities 1,13,14,16 . Thus, for a fixed mass or radius, the oxygen consumption rate per unit volume of non-luminal organoids is much higher than in spheroids, which often leads to a dead core -a recurring feature in these systems 17 . By combining computational models with images of mature brain organoid slices stained with a nuclear dye, Berger et al 17 recently identified a minimum threshold oxygen concentration for viability, Ccrit, of 0.04 mol/m 3 , which is also the critical value needed for mitochondrial ATP production 18 .…”

“…Thus, for a fixed mass or radius, the oxygen consumption rate per unit volume of non-luminal organoids is much higher than in spheroids, which often leads to a dead core -a recurring feature in these systems 17 . By combining computational models with images of mature brain organoid slices stained with a nuclear dye, Berger et al 17 recently identified a minimum threshold oxygen concentration for viability, Ccrit, of 0.04 mol/m 3 , which is also the critical value needed for mitochondrial ATP production 18 . The scientific community is aware of the urgent need to improve organoid technology in terms of quality and reproducibility of sample size and shape.…”