Mouse Wound Models and Preparation of Single-Cell Suspensions

Summary

This study presents two crucial experimental techniques: the construction of a murine wound model for assessing wound closure and the preparation of single-cell suspensions from murine skin to preserve cellular viability.

Abstract

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving diverse cell populations and the intricate wound microenvironment, complicates the assessment of treatment effects and their interactions using traditional experimental approaches. Single-cell transcriptome sequencing technology has revolutionized the understanding of cellular functions and mechanisms during wound healing. However, the variability in methodologies for creating mouse wound models introduces confounding factors that can impact experimental results. Furthermore, the process of dissociating mouse skin tissues presents significant technical hurdles, highlighting the critical need for high-quality single-cell suspensions for accurate transcriptome sequencing. Therefore, this study aims to optimize the construction of mouse wound models to accurately assess changes in wound closure while eliminating variables that could influence the subsequent sequencing result. Additionally, the preparation of single-cell suspensions was performed using both enzyme preparation protocols and test kit protocols to optimize cost and effectiveness.

Introduction

The mouse is a widely recognized animal model in research for its highly homologous genetic sequence to humans, rapid reproduction, cost-effectiveness, and manageable size¹. However, differences exist in the skin structure between mice and humans. Mice have a distinct cutaneous membrane layer that contributes to skin contraction, playing a crucial role in wound healing in this species. In contrast, human wound healing primarily involves re-epithelialization and granulation tissue formation¹.

Despite these interspecies differences in skin structure, the mouse model remains the preferred choice ....

Protocol

All procedures were approved by the Ethics Committee of the seventh Medical Center of Chinese PLA General Hospital, China. Male C57/BL6 mice aged 6 weeks were used. 

1. Mouse wound healing model

1. Animal grouping
   1. Randomly assign mice to experimental and control groups (6 each). House mice in a specific pathogen-free (SPF) animal facility, with a temperature maintained at 23-25 °C, humidity at 50%-60%, and a 12 h light-dark cycle.
   2. Prov.......

Discussion

Several crucial steps were essential for the protocol's success. These steps included precise preparation of the wound area by thorough shaving and cleaning, creating standardized wounds of specific size and depth, and diligent post-wound care to monitor infections and administer appropriate treatments. Standardization of these procedures ensured consistency across experimental conditions, thereby enhancing the reliability and reproducibility of results¹⁰.

We employed w.......

Materials

Name	Company	Catalog Number	Comments
75% Alcolchol	Liuhe	JIUJING500ML
C57/BL6J mice	Charles River Laboratories
Collagenase D + Dispase II	Roche	11088858001
Collagenase I	Roche	5349907103
Collagenase IV	Roche	5267439001
Cotton Swabs	Haishi	MIANQIAN
Dead Cell Removal Kit	Miltenyi Biotec	130-090-10
DNase I	Roche	10104159001
gentleMAC Dissociator	Miltenyi Biotec	130-093-235
Medical nylon thread	Jinlong	HM507
Multi Tissue Dissociation Kit 1	Miltenyi Biotec	130-110-201
punch biopsy needle	Integra Miltex
red blood cell lysis solution	Miltenyi Biotec	130-094-183
Surgical Scissor	Jinglu	JIANDAO
White petrolatum	Shangyuan	BAIFANSHILIN