Spatio-temporal dynamics enhance cellular diversity, neuronal function and further maturation of human cerebral organoids

Abstract

The bioengineerined and whole matured human brain organoids stand as highly valuable three-dimensional in vitro brain-mimetic models to recapitulate in vivo brain development, neurodevelopmental and neurodegenerative diseases. Various instructive signals affecting multiple biological processes including morphogenesis, developmental stages, cell fate transitions, cell migration, stem cell function and immune responses have been employed for generation of physiologically functional cerebral organoids. However, the current approaches for maturation require improvement for highly harvestable and functional cerebral organoids with reduced batch-to-batch variabilities. Here, we demonstrate two different engineering approaches, the rotating cell culture system (RCCS) microgravity bioreactor and a newly designed microfluidic platform (µ-platform) to improve harvestability, reproducibility and the survival of high-quality cerebral organoids and compare with those of traditional spinner and shaker systems. RCCS and µ-platform organoids have reached ideal sizes, approximately 95% harvestability, prolonged culture time with Ki-67 + /CD31 + /β-catenin+ proliferative, adhesive and endothelial-like cells and exhibited enriched cellular diversity (abundant neural/glial/ endothelial cell population), structural brain morphogenesis, further functional neuronal identities (glutamate secreting glutamatergic, GABAergic and hippocampal neurons) and synaptogenesis (presynaptic-postsynaptic interaction) during whole human brain development. Both organoids expressed CD11b + /IBA1 + microglia and MBP + /OLIG2 + oligodendrocytes at high levels as of day 60. RCCS and µ-platform organoids showing high levels of physiological fidelity a high level of physiological fidelity can serve as functional preclinical models to test new therapeutic regimens for neurological diseases and benefit from multiplexing.

Brain organoids: A revolutionary tool for modeling neurological disorders and development of therapeutics

“…One of the major challenges in generating brain organoids was the limited diffusion of nutrients and oxygen into the core, which leads to necrosis. Several types of bioreactors have been used to facilitate dynamic organoid culture and improve the survival of brain organoids by reducing cell death and necrotic zones (Figure 2a) (Goto‐Silva et al, 2019; Lancaster & Knoblich, 2014; Qian et al, 2016, 2018; Saglam‐Metiner et al, 2023; Silva et al, 2021). For example, cerebral organoids generated in a spinning bioreactor promoted the exchange of nutrients and oxygen, which improved the growth and development of organoids into defined regions of the brain (Lancaster & Knoblich, 2014).…”

“…Saglam et al (Saglam‐Metiner et al, 2023) used a microgravity bioreactor employing a horizontally rotating cell culture system (RCCS) to create an environment with laminar flow and microgravity conditions for reducing shear stress and cellular damage and increasing mass transfer. This setup enabled the generation of cerebral organoids by ensuring consistent and uniform hydrodynamic forces.…”

“…This setup enabled the generation of cerebral organoids by ensuring consistent and uniform hydrodynamic forces. The RCCS bioreactor led to precise control over cell‐cell interaction for long‐term culture, enhancing reproducibility and harvestability (Saglam‐Metiner et al, 2023). Brain organoids cultured in the RCCS bioreactor exhibited notable enhancements in neurogenesis and corticogenesis, resulting in a distinctive layered organization within brain organoids that distinguished them from shaker and spinner systems.…”

A Low‐Cost, User‐Friendly Rheo‐Optical Compression Assay to Measure Mechanical Properties of Cell Spheroids in Standard Cell Culture Plates

“…Additionally, it offers a straightforward and economical alternative for measuring spatio-temporal biophysical changes in biological samples, addressing a significant current demand. [38][39][40][41][42][43][44]…”

Dynamic culture of cerebral organoids using a pillar/perfusion plate for the assessment of developmental neurotoxicity

“…In addition, due to the prolonged cell culture required, the size of brain organoids often exceeds the diffusion limitation of nutrients and oxygen, leading to the formation of the necrotic core [27][28][29] . To address the diffusion issue, microphysiological systems (MPS) have been utilized for brain organoid culture, striving to replicate shear stress and blood flow as found in vivo, thereby enhancing organoid maturity and complexity [29][30][31][32][33] . Nonetheless, current microfluidic devices suffer from low throughput and user unfriendliness due to pumps and tubes required for operation.…”