Droplet 3D cryobioprinting for fabrication of free-standing and volumetric structures

Droplet-based bioprinting has shown remarkable potential in tissue engineering and regenerative medicine.However,it requires bioinks with low viscosities,which makes it challenging to create complex 3D structures and spatially pattern them with different materials.This study introduces a novel approach to bioprinting sophisti-cated volumetric objects by merging droplet-based bioprinting and cryobioprinting techniques.By leveraging the benefits of cryopreservation,we fabricated,for thefirst time,intricate,self-supporting cell-free or cell-laden structures with single or multiple materials in a simple droplet-based bioprinting process that is facilitated by depositing the droplets onto a cryoplate followed by crosslinking during revival.The feasibility of this approach is demonstrated by bioprinting several cell types,with cell viability increasing to 80%–90%after up to 2 or 3 weeks of culture.Furthermore,the applicational capabilities of this approach are showcased by bio-printing an endothelialized breast cancer model.The results indicate that merging droplet and cryogenic bioprinting complements current droplet-based bioprinting techniques and opens new avenues for the fabrication of volumetric objects with enhanced complexity and functionality,presenting exciting potential for biomedical applications.