Silk scaffolding drives self-assembly of functional and mature human brain organoids

Abstract

Human pluripotent stem cells (hPSCs) are intrinsically able to self-organize into cerebral organoids that mimic features of developing human brain tissue. These three-dimensional structures provide a unique opportunity to generate cytoarchitecture and cell-cell interactions reminiscent of human brain complexity in a dish. However, current in vitro brain organoid methodologies often result in intra-organoid variability, limiting their use in recapitulating later developmental stages as well as in disease modeling and drug discovery. In addition, cell stress and hypoxia resulting from long-term culture lead to incomplete maturation and cell death within the inner core. Here, we used a recombinant silk microfiber network as a scaffold to drive hPSCs to self-arrange into engineered cerebral organoids. Silk scaffolding promoted neuroectoderm formation and reduced heterogeneity of cellular organization within individual organoids. Bulk and single cell transcriptomics confirmed that silk cerebral organoids display more homogeneous and functionally mature neuronal properties than organoids grown in the absence of silk scaffold. Furthermore, oxygen sensing analysis showed that silk scaffolds create more favorable growth and differentiation conditions by facilitating the delivery of oxygen and nutrients. The silk scaffolding strategy appears to reduce intra-organoid variability and enhances self-organization into functionally mature human brain organoids.

From 2D to 3D: Development of Monolayer Dopaminergic Neuronal and Midbrain Organoid Cultures for Parkinson’s Disease Modeling and Regenerative Therapy

“…Another recent approach in tackling the necrotic core issue is the use of spider silk microfibers with laminin to assemble a hierarchical 3D scaffold for iPSCs to self-arrange into MOs. The silk scaffolding forms microcavity networks that increase cell surface to volume ratio that facilitates oxygen flow and metabolic waste removal that result in a reduction in necrosis [253]. Similarly, others have used carbon fibers as a novel scaffold for MO generation to promote neuronal survival [199].…”

Advanced Technologies for Studying Microbiome–Female Reproductive Tract Interactions: Organoids, Organoids-on-a-Chip, and Beyond

“…17,90 Creating stable and reproducible oxygen gradients across organoid cultures presents a technical challenge, but recent advances such as microfluidic organoid culture devices and oxygen-permeable scaffolds offer promising tools for maintaining controlled oxygen levels. 78,93 The human-microbial crosstalk module (HuMiX) represents a significant advancement in this field, allowing anaerobic bacteria to be maintained in an almost anoxic compartment. 94 The anoxic environment is created by perfusing an anoxic medium.…”

“…17 90 Creating stable and reproducible oxygen gradients across organoid cultures presents a technical challenge, but recent advances such as microfluidic organoid culture devices and oxygen-permeable scaffolds offer promising tools for maintaining controlled oxygen levels. 78 93…”

Improved Protocol for Reproducible Human Cortical Organoids Reveals Early Alterations in Metabolism withMAPTMutations

“…19,[38][39][40][41] Several studies have optimized protocols to improve uniformity between cerebral organoids and between batches. 4,6,[42][43][44] Most were developed using scRNA-seq analysis of 1-5 lines to assess cell composition. However, hPSC lines can differ considerably, both donor-to-donor and between lines of the same donor.…”