Engineered gelatin hydrogels as a well-defined, tunable substrate for cortical brain organoid models

Introduction:: The human brain is composed of diverse cell types arranged in a delicate architecture. Recapitulating this diversity is fundamental for studies of neurodevelopment, neurodegeneration, and therapeutic intervention. Cortical brain organoids derived from induced pluripotent stem cells have been widely recognized as scalable, representative models of human neural phenotypes. However, many organoid differentiation protocols rely on Matrigel – a biomaterial secreted by mouse sarcoma cells. Matrigel is poorly tunable, chemically undefined, has batch-to-batch heterogeneity, and may contain xenobiotics; these deficiencies have led to interest in exploring synthetic or semi-synthetic alternatives for cell culture applications. In addition to overcoming the limitations of Matrigel, synthetic matrices also enable precise control of instructive cell – extracellular matrix interactions, which may aid organoid development and maturation. Thus, we sought to develop an engineered matrix that could replace or surpass Matrigel to support cortical organoid culture.


Materials and Methods:: To create a well-defined matrix for organoid growth, a gelatin backbone was functionalized with methacrylate groups to facilitate photo-initiated polymerization. The resulting methacrylated gelatin (GelMA) was then functionalized with an epitope of the extracellular domain of the human neural cadherin (N-cadherin) protein (Figure A), as our previous work has demonstrated this peptide can support neural cell adhesion, survival, and maturation in 3D culture. Matrix properties were modulated by adjusting crosslinking time, crosslinker concentration, matrix concentration, crosslinking modality, and N-cadherin peptide concentration. As controls, unmodified gelatin and GelMA hydrogels, as well as Matrigel hydrogels, were used. Cortical organoids were embedded in these matrices, and then differentiated for 30 – 60 days.


Results, Conclusions, and Discussions:: Methacrylate and peptide conjugation reactions were confirmed using ¹H NMR spectroscopy (Figure B). After polymerization, scanning electron microscopy was used to measure pore sizes ranging between 10 - 100 μm. Young’s moduli, as assessed by atomic force microscopy, was tunable between 0.3 kPa and 4.7 kPa.

Extracellular matrix stiffness and peptide conjugation both had a strong influence on cortical organoid morphology. In softer hydrogel conditions (~0.3 kPa), organoids had larger diameters (p < 0.001) and were less circular (p < 0.001) compared to organoids cultured in stiffer conditions (~2.6 kPa). Moreover, this effect was independent of crosslinking modality (enzymatic crosslinking vs. UV photopolymerization). Most strikingly, organoids grown in soft gels reproducibly yielded extensive beta three tubulin (TUBB3+) neurite extensions. Interestingly, the addition of conjugated peptide was also able to produce extensive TUBB3+ neurites at stiffer hydrogel conditions (Figure C). Thicker layers of TUBB3+ cells formed in stiffer GelMA-based hydrogels (p < 0.001), suggesting these may be more optimal for organoid generation (Figure C). Furthermore, growth in the gelatin-based hydrogels was more constrained compared to Matrigel, which might aid in nutrient diffusion and yield healthier organoids (Figure E).

Importantly, canonical markers of neural development were present in day 60 organoids (low levels of stemness marker SOX2; high expression of layer-specific neural markers CTIP2, SATB2) (Figure F). Transcriptomic analysis of day 60 organoids using scRNAseq is undergoing and will robustly characterize the milieu of cell fates in cortical organoids in peptide-functionalized gelatin relative to Matrigel. Taken together, these data suggest that the engineered gelatin hydrogels are a viable substrate for the support of cortical organoids.

By engineering a custom biomaterial for cortical organoid differentiation, we were able to investigate the role of extracellular matrix cues on cell fate. Moreover, this novel biomaterial may be a viable replacement for Matrigel in typical brain organoid workflows. Circumventing the need for Matrigel in cerebral organoid culture is an important advancement in the standardization and scalability of organoid culture. Our approach could also be leveraged to design other bio-instructive hydrogels conjugated with peptide motifs to further enhance organoid models for the brain and other lineages.


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