ABSTRACT
Hematopoietic stem cells (HSCs) are the apical cells of the hematopoietic system, giving rise to cells of the blood and lymph lineages. HSCs reside primarily within bone marrow niches that contain matrix and cell-derived signals that help inform stem cell fate. Aspects of the bone marrow microenvironment have been captured in vitro by encapsulating cells within hydrogel matrices that mimic native mechanical and biochemical properties. Hydrogel microparticles, or microgels, are increasingly being used to assemble granular biomaterials for cell culture and noninvasive delivery applications. Here, we report the optimization of a gelatin maleimide hydrogel system to create monodisperse gelatin microgels via a flow-focusing microfluidic process. We report characteristic hydrogel stiffness, stability, and swelling characteristics as well as encapsulation of murine hematopoietic stem and progenitor cells, and mesenchymal stem cells within microgels. Microgels support cell viability, confirming compatibility of the microfluidic encapsulation process with these sensitive bone marrow cell populations. Overall, this work presents a microgel-based gelatin maleimide hydrogel as a foundation for future development of a multicellular artificial bone marrow culture system.
ABSTRACT
BACKGROUND: Polyphenol catechins from green tea, particularly (-)-epigallocatechin-3-gallate (EGCG), exhibits numerous beneficial health effects, although the mechanisms remain unclear. METHODS: In this study, the mechanism of EGCG-mediated healing in an experimentally injured zebrafish model was examined at the cellular and molecular level using confocal microscopy and gene expression analysis. RESULTS: The mechanisms of action of EGCG were shown to involve: (1) reducing neutrophil response (accumulation, travel speed, and distance) and (2) downregulating the expression of IL-1ß, TNFα, and related signaling pathways. As determined by dynamic time-lapse tracking studies, the local accumulation of neutrophils with high migration speeds after wounding (n=33 cells, v=0.020 µm/s, d=37.8 µm), underwent significant reduction following treatment with EGCG doses of 300 µM (n=22 cells, v=0.013 µm/s, d=39.5 µm) and 600 µM (n=18 cells, v=0.008 µm/s, d=9.53 µm). Reverse transcription polymerase chain reaction studies revealed that several signature genes in the IL-1ß, TNFα, and related signaling pathways were downregulated after EGCG treatment. CONCLUSION: The convenience, transparency, and simplicity of the zebrafish model facilitate tracking of fluorescent neutrophils in real time, in order to monitor inflammation, and assess the impact of therapeutic agents.
