Human brain tissue models such as cerebral organoids are essential tools for developmental and biomedical research. Current methods to generate cerebral organoids often utilize Matrigel as an external scaffold to provide structure and biologically relevant signals. Matrigel however is a nonspecific hydrogel of mouse tumor origin and does not represent the complexity of the brain protein environment. In this study, we investigated the application of a decellularized adult porcine brain extracellular matrix (B-ECM) which could be processed into a hydrogel (B-ECM hydrogel) to be used as a scaffold for human embryonic stem cell (hESC)-derived brain organoids. We decellularized pig brains with a novel detergent- and enzyme-based method and analyzed the biomaterial properties, including protein composition and content, DNA content, mechanical characteristics, surface structure, and antigen presence. Then, we compared the growth of human brain organoid models with the B-ECM hydrogel or Matrigel controls in vitro. We found that the native brain source material was successfully decellularized with little remaining DNA content, while Mass Spectrometry (MS) showed the loss of several brain-specific proteins, while mainly different collagen types remained in the B-ECM. Rheological results revealed stable hydrogel formation, starting from B-ECM hydrogel concentrations of 5 mg/mL. hESCs cultured in B-ECM hydrogels showed gene expression and differentiation outcomes similar to those grown in Matrigel. These results indicate that B-ECM hydrogels can be used as an alternative scaffold for human cerebral organoid formation, and may be further optimized for improved organoid growth by further improving protein retention other than collagen after decellularization.

Brain organoids are considered to be a highly promising in vitro model for the study of the human brain and, despite their various shortcomings, have already been used widely in neurobiological studies. Especially for drug screening applications, a highly reproducible protocol with simple tissue culture steps and consistent output, is required. Here we present an engineering approach that addresses several existing shortcomings of brain organoids. By culturing brain organoids with a polycaprolactone scaffold, we were able to modify their shape into a flat morphology. Engineered flat brain organoids (efBOs) possess advantageous diffusion conditions and thus their tissue is better supplied with oxygen and nutrients, preventing the formation of a necrotic tissue core. Moreover, the efBO protocol is highly simplified and allows to customize the organoid size directly from the start. By seeding cells onto 12 by 12 mm scaffolds, the brain organoid size can be significantly increased. In addition, we were able to observe folding reminiscent of gyrification around day 20, which was self-generated by the tissue. To our knowledge, this is the first study that reports intrinsically caused gyrification of neuronal tissue in vitro. We consider our efBO protocol as a next step towards the generation of a stable and reliable human brain model for drug screening applications and spatial patterning experiments.

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