Three-Dimensional Culture Decreases the Angiogenic Ability of Mouse Macrophages.

Haoxin Shi,Qing Shi,Miao Qin,Yuwei Tan,Zhenxia Han,Zengjun Li,Dong Li,Xiaodong Mu

doi:10.3389/fimmu.2021.795066

Abstract

Macrophages play important roles in angiogenesis; however, previous studies on macrophage angiogenesis have focused on traditional 2D cultures. In this study, we established a 3D culture system for macrophages using collagen microcarriers and assessed the effect of 3D culture on their angiogenic capabilities. Macrophages grown in 3D culture displayed a significantly different morphology and arrangement under electron microscopy compared to those grown in 2D culture. Tube formation assays and chick embryo chorioallantoic membrane assays further revealed that 3D-cultured macrophages were less angiogenic than those in 2D culture. Whole-transcriptome sequencing showed that nearly 40% of genes were significantly differently expressed, including nine important angiogenic factors of which seven had been downregulated. In addition, the expression of almost all genes related to two important angiogenic pathways was decreased in 3D-cultured macrophages, including the two key angiogenic factors, VEGFA and ANG2. Together, the findings of our study improve our understanding of angiogenesis and 3D macrophage culture in tissues, and provide new avenues and methods for future research on macrophages.

Highlights

Macrophages are a type of immune cell that are widely distributed in all bodily tissues and play vital roles in development, homeostasis, tissue repair, and immunity [1]
In order to explore the effect of 3D culture on the morphology of macrophages, we used scanning electron microscopy to observe two groups of macrophages in 3D and 2D culture
We found that the growth rate of cells under 3D culture was slower; we first detected the cell cycle of the macrophages

Summary

Introduction

Macrophages are a type of immune cell that are widely distributed in all bodily tissues and play vital roles in development, homeostasis, tissue repair, and immunity [1]. Macrophages play a vital role in promoting angiogenesis by secreting various pro-angiogenetic cytokines and growth factors, such as Traditional macrophage research has mostly been based on two-dimensional (2D) culture, in which cells grow in a monolayer under adherent conditions. This does not accurately simulate the environment of the cells in 3D structures in vivo, including cell-cell and cell-extracellular matrix (ECM) interactions [5]. Compared to cells grown in a 2D environment, those in 3D culture have no polarity, discrete matrix fibers, and variable stiffness These cells adhere in three dimensions and their diffusion and migration are impeded [9,10,11]. The enhanced contact between cells increases intercellular signaling, facilitates developmental processes, and allows cells to differentiate into more complex structures than through traditional culture methods [4, 12]

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