Title: Engineering 3-D Neural Organoid Morphology
Legend: (A) Schematic of micro-injection molding process to create poly(vinyl alcohol)-calcium (PVOH-Ca) composites that can sever as a water-soluble, sacrificial template to mold morphologically complex, microscale cavities within 3-D hydrogels. (B) Representative images of micro-injection molded 10% (w/w) PVOH-Ca(C2H3O2)2 sacrificial fiber templates (i), the molded channel within an alginate hydrogel (ii), and a profile (iii) and cross-sectional (iv) view of the microCT-imaged channel geometry. (C) Experimental timeline of neuroepithelial organoid derivation. Alginate hydrogels with molded cavities were prepared the day before hESC injection. (B) Brightfield image of organoids after 16 days of culture demonstrating their elliptical morphology. (D) Immunocytochemistry of neuroepithelial organoid cryosections for basement membrane protein Laminin (Lam) and NEC polarization marker N-cadherin (Ncad) after 4, 8, and 16 days of bioreactor culture. Scale bars are (B) 3 mm, (C) 500 μm, (D) 100 μm.
Citation: J.D. McNulty, C. Marti-Figueroa, F. Seipel, J.Z. Plantz, T. Ellingham, L.J.L. Duddleston, et al., Micro-injection molded, poly(vinyl alcohol)-calcium salt templates for precise customization of 3D hydrogel internal architecture, Acta Biomaterialia. 95 (2019) 258–268. doi:10.1016/j.actbio.2019.04.050.
Abstract: In tissue engineering applications, sacrificial molding of hydrogel monoliths is a versatile technique for creating 3D molds to control tissue morphology. Previous sacrificial templates fabricated by serial processes such as solvent casting and thermal extrusion/fiber drawing can be used to effectively mold internal geometries within rapidly polymerizing, bulk curing hydrogels. However, they display poorer performance in controlling the geometry of diffusion limited, ionically cross-linked hydrogels, such as alginate. Here, we describe the use of poly(vinyl alcohol)-calcium salt templates (PVOH-Ca) fabricated by micro-injection molding, a parallel mass-production process, to conveniently cast internal geometries within both bulk curing hydrogels and ionically cross-linked alginate hydrogels. Calcium salt solubility was discovered to be a critical factor in optimizing the polymer composite’s manufacturability, mechanical properties, and the quantity of calcium released upon template dissolution. Metrological and computed tomography (CT) analysis showed that the template’s calcium release enables precise casting of microscale channel geometries within alginate hydrogels (6.4 ± 7.2% average error). Assembly of modular PVOH-Ca templates to mold 3D channel networks within alginate hydrogels is presented to demonstrate engineering scalability. Moreover, the platform is used to create hydrogel molds for engineering human embryonic stem cell (hESC)-derived neuroepithelial organoids of a microscale, biomimetic cylindrical morphology. Thus, injection molded PVOH-Ca templates facilitate customization of hydrogel sacrificial molding, which can be used to generate 3D hydrogels with complex internal microscale architecture for diverse tissue engineering applications.
About the Lab: The goal of the Ashton lab is (1) to understand, model, and recapitulate in vitro the instructive signals utilized by human embryos to pattern tissue-specific differentiation and morphogenesis of pluripotent stem cells and (2) apply this knowledge towards the rational design of tissue engineered scaffolds, neural organoid tissues, and other regenerative therapeutic strategies. Currently, the lab primarily focus on generating tissues and therapies for the central nervous system.