Neutrophil Granulopoiesis optimized through Ex vivo Expansion of Hematopoietic Progenitors in Engineered 3D Gelatin Methacrylate Hydrogels.

Neutrophils are the first line of defense of the innate immune system. In response to methicillin resistant Staphylococcus aureus infection in skin, hematopoietic stem and progenitor cells (HSPCs) traffic to wounds and undergo extramedullary granulopoiesis, which produces neutrophils necessary to resolve infection. This prompted engineering a gelatin methacrylate (GelMA) hydrogel that encapsulates HSPCs within a matrix amenable to subcutaneous delivery. We studied the influence of hydrogel mechanical properties to produce an artificial niche for granulocyte-monocyte progenitors (GMPs) to efficiently expand into functional neutrophils that can populate infected tissue. Lin-cKIT+ HSPCs, harvested from LysM-EGFP neutrophil reporter mice, were encapsulated in GelMA hydrogels of varying polymer concentration and UV-crosslinked to produce HSPC-laden gel of specific stiffness and mesh sizes. Softer 5% GelMA gels yielded the most viable progenitors and effective cell-matrix interactions. Compared to suspension culture, 5% GelMA resulted in a two-fold expansion of mature neutrophils that retain antimicrobial functions including degranulation, phagocytosis, and ROS production. When implanted dermally in C57BL/6J mice, luciferase-expressing neutrophils expanded in GelMA hydrogels were visualized at the site of implantation for over 5 days. We demonstrate the potential of GelMA as an artificial niche for delivering HSPCs directly to the site of skin infection to promote local granulopoiesis. This article is protected by copyright. All rights reserved.