‘NextGen’ human brain organoids using 3D printed gelatin methacrylate

Tomaskovic-Crook E1,2, Zhang B1, Bourke JL3,4, Gu Q1, Kapsa RM3, Wallace GG1 and Crook JM1,2,5

  1. ARC Centre of Excellence for Electromaterials Science (ACES), AIIM, University of Wollongong, Innovation Campus, North Wollongong, NSW.
  2. IHMRI, University of Wollongong, Wollongong, NSW.
  3. ACES, St Vincent’s Hospital, University of Melbourne, Fitzroy, VIC.
  4. Dept Medicine, St Vincent’s Hospital, University of Melbourne, Fitzroy, VIC.
  5. Dept Surgery, St Vincent’s Hospital, University of Melbourne, Fitzroy, VIC.

The generation of brain organoids derived from human pluripotent stem cells (PSCs) is a significant step towards better in vitro modelling of neurodevelopment and disease. Brain organoids are discerned by their cellular and structural complexity, with characteristics of developing embryonic brains. Conventional methods of organoid formation are limited to small-scale operation and require multiple handling steps following stem cell aggregation, coating with expensive and undefined tumour-derived Matrigel™ basement membrane preparation, and arduous bioreactor based differentiation and expansion methods. We have initially demonstrated gelatin methacrylate (GelMA) to be a cell growth substrate for rapid and novel induction of brain organoids from human induced PSCs (iPSCs). GelMA is a versatile, 3D printable semisynthetic matrix that incorporates the intrinsic bioactivity of natural matrices with the fidelity of synthetic biomaterials for more defined and clinically-compliant cell support. Towards scaling up organoid production we have now 3D printed GelMA-based microwell arrays to generate large numbers of organoids for higher throughput R&D. Constructs consist of densely packed cell soma with regional divisions resembling cortical plate or rudimentary grey matter tissue with underlying white matter-like tissue, as well as hollow neural tube-like structures. With larger numbers of organoids we are progressing our understanding through immunofluorescent-based histochemistry of cortical lamination using layer-specific markers of cerebral neocortex and early progenitor regions; markers including RELN, CTIP2, TBR1, SATB2, PAX6, NES, and SOX2, as well as the forebrain specific marker, FOXG1. Moreover, we have demonstrated coordinated glutamate-responsive neural network activity of formed neurons by extracellular recordings using multi-electrode arrays (MEAs). The optimised ’NextGen’ method provides a defined, simplified and higher throughput platform for ’brain on a bench’ research and translation of iPSCs, neural derivatives and neural organoids, including in vitro modelling of brain development and disease, tissue engineering and regenerative medicine.