Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells

Summary

This study developed an optimized protocol for the isolation of murine mesangial cells (MCs) and their ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression.

Abstract

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for isolating, purifying, and culturing glomerular MCs have been developed and optimized since the 1980s for use in biomedical research, particularly in the field of nephrology. Mice are the most frequently used experimental animal models in research on renal diseases. In this study, we developed an optimized protocol for murine MCs isolation and ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression. This optimized approach significantly reduces the study duration for researchers and enables long-term cell preservation. The necessary equipment for the procedures is easily accessible in a basic biomedical laboratory, and the procedural steps are straightforward. The acquisition of target cells requires only 2-3 weeks, a reduction of at least 1 week compared to existing methods.

Introduction

The glomerulus is a network of capillaries that performs the essential task of filtering blood to form urine¹. Mesangial cells (MCs) are embedded within the mesangial matrix, situated between the glomerular capillaries, and are uniquely positioned to influence glomerular dynamics through their diverse functions². MCs play crucial roles in the glomerulus, including glomerular development, structural support for glomerular capillaries, phagocytosis, and the production of the glomerular basement membrane matrix³. The study of mesangial cells is pivotal for advancing our understanding of renal physiol....

Protocol

Animal experiments complied with the ARRIVE guidelines, and all animal procedures were conducted in accordance with national legislation and the European Commission Directive (2010/63/EU). Mice were housed and maintained in pathogen-free conditions, in compliance with the requirements of the Animal Care and Use Committees of West China Hospital, Sichuan University. All experimental animal studies were approved by the Animal Care and Use Committees of West China Hospital, Sichuan University. Eight-week-old C57BL/6JGpt male mice were used for mesangial cell isolation in this assay. The details of the reagents and equipment used in this study are listed in the Ta....

Results

This study developed an optimized protocol for murine MCs isolation and ex vivo cell culture. To our knowledge, no standard method exists for validating primary MCs ex vivo. According to previous publications, MCs are characterized by the expression of αSMA, Vimentin, and Fibronectin^3,14,15. Western blot analysis was used to detect the expression levels of MCs. The MCs isolated in this study exhibited significa.......

Discussion

Kidney-related diseases are highly prevalent, and mice are widely used as the primary animal model for studying these conditions due to their genetic similarity to humans and the availability of well-established disease models. Mesangial cells play a crucial role in maintaining the normal structure and function of the glomerulus by providing structural support, regulating glomerular filtration, and participating in immune responses. While various experimental techniques exist for isolating mesangial cells from humans, ra.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 µm Cell Strainer	Biosharp	BS-100-CS
100 mm Petri Dish	Sorfa	230301
15 mL Centrifuge Tube	Sorfa	411000
15 mm glass bottom cell culture dish	Sorfa	201200
180 kDa Plus Prestained Protein Marker	Vazyme	MP201-01
2 mM L-glutamine	BasalMedia	S210JV
4% Paraformaldehyde	Biosharp	BL539A
40 µm Cell Strainer	Biosharp	BS-40-XBS
50 mL Centrifuge Tube	Sorfa	41000
60 mm Petri Dish	Sorfa	230201
75% Alcohol	Knowles	64-17-5
96 Well Cell Culture Plates, TC-treated	Servicebio	CCP-96H
Antibiotics (10 μg/mL ceftriaxone plus 100 μg/mL gentamicin)	NCM	C100C5
BCA (Bicinchoninic acid) Protein Assay kit	CWBIO	CW0014S
Cell Counting Kit-8	Oriscience	CB101
Confocal Microscope	FV-3000	Olympus
DMSO	Sigma	D2650
DMSO	Sigma	D2650
Earle's Balanced Salt Solution (1x EBSS)	Beyotime	C0213
Fetal Bovine Serum (FBS)	Excell	FSP500
Fluorescence Cell Analyzer	Mira FL	Countstar
Fluoromount media	Southern Biotech	0100-01
Insulin, Transferrin, Selenium Solution 100x (ITS -G)	Gibco	41400045
Inverted Fluorescence Microscope	Olympus	IX83
Lysis Buffer	Adilab	PP1101
One-Step PAGE Preparation Kit (10%)	Oriscience	PB102
One-Step PAGE Preparation Kit (7.5%)	Oriscience	PB101
PE anti-mouse CD140b Antibody	Biolegend	323605
Phosphate Buffered Saline (PBS)	Servicebio	G4202
Plastic Cell Pestle	Biofil	CC-4090
Proteinase Inhibitor Cocktail	Roche	4693159001
PVDF Membrane	Vazyme	E802-01
Recombinant Human Insulin	Solarbio	11061-68-0
Roswell Park Memorial Institute (RPMI) 1640 Medium	Corning	10-040-CV
SDS-PAGE Sample Loading Buffer (5x)	Servicebio	G2013
Specially customized Roswell Park Memorial Institute (RPMI) 1640 Medium (D-valine instead of L-valine)	Procell	WH3923U222
TBS (Tris Buffered Saline)	Servicebio	G0001-2L
Triton X-100	Biosharp	BS084
Trypsin-EDTA (0.25%)	Gibco	25200072
Tween 20	Biosharp	BS100
Type I collagenase	Solarbio	CB140