A Flexible Fibrin‐Based Platform for Driving One‐Day Self‐Assembly of Millimeter‐Sized Cell‐Rich Spheroids and Their Applications: An Old Material with New Tricks

Abstract Cell‐rich spheroids have gained increasing applications due to their ability to mimic the 3D microenvironment of real tissues. Given human organ dimensions, millimeter‐sized (>1 mm) spheroids are more clinically valuable than micro‐sized ones (<500 µm). However, constructing millimeter‐sized spheroids faces the primary challenge of core necrosis due to insufficient nutrient diffusion during long‐term in vitro cultivation (>3 days). Developing rapid spheroid fabrication strategies can address the issue but remains challenging. Here, a flexible fibrin‐based platform for rapid fabrication of the spheroids is developed using a one‐step manufacturing technology. Cell‐laden fibrin hydrogels are polymerized from cell‐containing fibrinogen by thrombin‐catalyzed reactions within minutes and cultured in an agarose‐coated plate. Benefiting from the unique viscoelasticity, softness, and cell adhesion natures of fibrin, the expression levels of several cellular junction proteins are significantly upregulated compared to the scaffold‐free spheroids. As a result, cells rapidly self‐assemble into 1–15 mm‐sized spheroids within 6–24 h, accommodating high cell density (6 × 10 4 cells µL −1 ) without leakage. The spheroid formation platform is also flexible for various cell densities and cell types. Moreover, the spheroids effectively mimic the 3D tissue niches, showing promising potential in tumor drug screening with MCF‐7 spheroids and liver regeneration with HepG2 spheroids.