Advancing Organ-on-Chip Models With a Sacrificial Granular Hydrogel Strategy for Enhanced Permeability and Biomimicry.

Infectious diseases such as malaria, leishmaniasis, and human immunodeficiency virus (HIV) involve pathogens with complex life cycles that span multiple organs, including the bone marrow (BM), a niche for latent or cryptic infections. Studying these hidden stages in patients presents significant technical and ethical challenges, underscoring the need for advanced in vitro models such as organ-on-chip (OoC) platforms. While cell-laden hydrogels can replicate tissue-like 3D-microenvironments, their small mesh size may restrict pathogen migration and cell-pathogen interactions, both critical for establishing infection on-chip. To overcome this limitation, this work develops a "reversed" granular hydrogel strategy that creates interconnected microporosity in hydrogels incorporated into organ-on-chip compartments. Sacrificial alginate (ALG) µgels are embedded as porogens in a fibrin-collagen (FIB-COL) precursor inside a custom BM-on-chip and, after crosslinking, are selectively removed by in situ enzymatic/chemical leaching to yield highly porous hydrogels (pFIB-COL). The pFIB-COL supports 3D-cultures of mesenchymal stromal cells, endothelial cells, and erythroblasts. Physical and cellular analyses show reduced flow resistance, enhanced particle and cell permeation, more uniform cell distribution and improved endothelial network formation compared with native FIB-COL. This versatile strategy is readily adaptable to other hydrogel systems, providing a valuable tool for the faithful modeling of infection processes in biomimetic 3D-microenvironments within OoC devices.