Effects of substrate stiffness on the polarization states of murine bone marrow-derived neutrophils

The substrate stiffness plays an important role in mediating the cellular behavior. Neutrophils predominate the early inflammatory response and initiate the regeneration. The neutrophil activation can be regulated by physical cues. However, it is not known how neutrophils respond to substrate stiffness, which is of significant importance in determining the outcomes of engineered tissue mimics. Herein, a three-dimensional culture system made of hydrogel for bone marrow-derived neutrophils was developed to explore the effects of varying stiffness (1.5, 2.6, and 5.7 kPa) on neutrophil phenotype and polarization states. Murine bone marrow-derived neutrophils were encapsuled and cultured in GelMA hydrogel with different stiffnesses (1.5, 2.6, and 5.7 kPa) for 2 days. Then the hydrogel was lysed and released the cells, which were collected. RNA was extracted and differential gene expression was examined by RNAsequencing. Three experimental groups were set up in this study accoding to the substrate stiffness: Low (1.5kpa), middle (2.6kpa), high (5.7kpa). The group Low was set as control. Three replicates of RNA samples per experimental group were analyzed.

底物刚度在调控细胞行为中发挥关键作用。中性粒细胞（Neutrophils）主导早期炎症应答并启动组织再生，其活化可受物理信号调控。然而，目前尚不明确中性粒细胞如何响应底物刚度，而该问题对于预测工程化组织模拟物的结局具有重要意义。本研究构建了一种基于水凝胶（hydrogel）的三维培养体系，用于培养骨髓来源中性粒细胞，以探究不同刚度（1.5、2.6、5.7 kPa）对中性粒细胞表型及极化状态的影响。将小鼠骨髓来源中性粒细胞包埋于不同刚度（1.5、2.6、5.7 kPa）的明胶甲基丙烯酸酯水凝胶（GelMA）中培养2天，随后裂解水凝胶以释放并收集细胞，提取总RNA并通过RNA测序（RNAsequencing）分析差异基因表达。本研究根据底物刚度设置三组实验组：低刚度组（1.5 kPa）、中刚度组（2.6 kPa）与高刚度组（5.7 kPa），其中低刚度组作为对照。每个实验组设置三份RNA样本重复进行分析。