Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures

Abstract

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape.

Recent Advances in Multi‐Photon 3D Laser Printing: Active Materials and Applications

“…In a follow-up study in 2023, the authors presented a detailed study of organoid development in a confined space of the same material presenting data on organotypic spinal cord, lung cancer organoids, liver organoids, and organotypic culture of lung epithelium rudiments. [216] In this work, Urciulo et al used a basement membrane extracellular matrix for encapsulation of the organoids and added the previously described HCCfunctionalized photoresist on top of the encapsulation gel. [216] After incubation, the authors were able to print solid structures inside of the gel.…”

“…[216] In this work, Urciulo et al used a basement membrane extracellular matrix for encapsulation of the organoids and added the previously described HCCfunctionalized photoresist on top of the encapsulation gel. [216] After incubation, the authors were able to print solid structures inside of the gel. Subsequent removal of the basement membrane extracellular matrix led to the printed HCC-gelatin structure with encapsulated organoids.…”

Diffusion‐Based 3D Bioprinting Strategies

“…This combined inward-outward diffusion approach enables dual-material constructs to be generated dynamically during culture, facilitating studies of the effect of matrix architecture and mechanical properties on cell behavior. [77] Due to their ability to integrate multiple biopolymers into a single construct, diffusion-based bioprinting strategies open up opportunities for the customization of bioinks for specific cell types. We anticipate that these strategies will be generalized to-ward new materials and crosslinking chemistries, thus expanding the library of compatible bioinks for creating more heterogeneous and biofunctional constructs.…”

“…[76] As applied previously with the inward diffusion of crosslinkers coupled with the outward diffusion of a temporary viscosity enhancer, [57] a combined inward-outward diffusion approach has more recently been employed for tailoring cell-and organoid-laden bioprinted constructs. [77] Such strategies will enable the fabrication of constructs with enhanced biological functionality and geometric complexity. Furthermore, control over the mechanical properties of printed constructs can be improved by utilizing a wider library of crosslinkers, adjusting the crosslinker concentration and diffusion time, and applying secondary crosslinking steps.…”

“…This combined inward‐outward diffusion approach enables dual‐material constructs to be generated dynamically during culture, facilitating studies of the effect of matrix architecture and mechanical properties on cell behavior. [ 77 ]…”