3D co-culture of macrophages and fibroblasts in a sessile drop array for unveiling the role of macrophages in skin wound-healing

Xiaoyan Lyu; Feiyun Cui; Hang Zhou; Bo Cao; Xiaolan Zhang; Minghui Cai; Shulong Yang; Bangyong Sun; Gang Li

doi:10.1016/j.bios.2023.115111

3D co-culture of macrophages and fibroblasts in a sessile drop array for unveiling the role of macrophages in skin wound-healing

Biosens Bioelectron. 2023 Apr 1:225:115111. doi: 10.1016/j.bios.2023.115111. Epub 2023 Jan 28.

Authors

Xiaoyan Lyu¹, Feiyun Cui², Hang Zhou³, Bo Cao², Xiaolan Zhang², Minghui Cai², Shulong Yang⁴, Bangyong Sun⁵, Gang Li⁶

Affiliations

¹ Department of Dermatology, Laboratory of Dermatology, Clinical Institute of Inflammation and Immunology, Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu, 610041, China. Electronic address: lxiaoyan@scu.edu.cn.
² School of Basic Medical Sciences, Harbin Medical University, Harbin, 150081, China.
³ The Ministry of Education Key Laboratory of Clinical Diagnostics, School of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
⁴ Department of Pediatric Surgery, The Sixth Affiliated Hospital of Harbin Medical University, Harbin, 150081, China.
⁵ Key Laboratory of Optoelectronic Technology and Systems, Ministry of Education, Defense Key Disciplines Lab of Novel Micro-Nano Devices and System Technology, Chongqing University, Chongqing, 400044, China.
⁶ Key Laboratory of Optoelectronic Technology and Systems, Ministry of Education, Defense Key Disciplines Lab of Novel Micro-Nano Devices and System Technology, Chongqing University, Chongqing, 400044, China. Electronic address: gang_li@cqu.edu.cn.

PMID: 36731395
DOI: 10.1016/j.bios.2023.115111

Abstract

Three-dimensional (3D) heterotypic multicellular spheroid models play important roles in researches of the proliferation and remodeling phases in wound healing. This study aimed to develop a sessile drop array to cultivate 3D spheroids and simulate wound healing stage in vitro using NIH-3T3 fibroblasts and M2-type macrophages. By the aid of the offset of surface tension and gravity, the sessile drop array is able to transfer cell suspensions to spheroids via the superhydrophobic surface of each microwell. Meanwhile, each microwell has a cylinder hole at its bottom that provides adequate oxygen to the spheroid. It demonstrated that the NIH-3T3 fibroblast spheroid and the 3T3 fibroblast/M2-type macrophage heterotypic multicellular spheroid can form and maintain physiological activities within nine days. In order to further investigate the structure without destroying the entire spheroid, we reconstructed its 3D morphology using transparent processing technology and the Z-stack function of confocal microscopy. Additionally, a nano antibody-based 3D immunostaining assay was used to analyze the proliferation and differentiation characteristics of these cells. It found that M2-type macrophages were capable of promoting the differentiation of 3T3 fibroblast spheroid. In this study, a novel, inexpensive platform was constructed for developing spheroids, as well as a 3D immunofluorescence method for investigating the macrophage-associated wound healing microenvironment.

Keywords: Macrophage modulated fibroblasts; Sessile drop array; Three-dimensional cell co-culture; Wound healing microenvironment.

MeSH terms

Biosensing Techniques*
Coculture Techniques
Fibroblasts
Macrophages
Spheroids, Cellular