In vitro Isolation and Culturing of Mouse Primary Chondrocytes for Cartilage Biology

Summary

This protocol enables the isolation of sufficient primary chondrocytes within 6-8 h, facilitating further in-depth investigations of cartilage biology and cartilage disease mechanisms.

Abstract

Articular cartilage destruction leads to altered chondrocyte activity, which is a major causative factor in a variety of cartilage diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). As the most abundant cell type in cartilage, an in-depth study of the biological properties of chondrocytes is essential for the development of effective disease treatment strategies. Isolation and culturing of primary chondrocytes provide important experimental materials for the investigation of cartilage disease and contribute to unraveling the mechanisms of cartilage injury and repair. This protocol provides a detailed description of the in vitro isolation and culturing methods for primary chondrocytes derived from mice. By using the optimized collagenase II protocol, chondrocyte isolation can be completed within 8 h for neonatal mouse knee joint cartilage specimens. The cell yield is approximately 1-2 × 10³ cells/mg of cartilage tissue, varying with tissue freshness and initial cell density; the isolated cells exhibit >90% viability. This protocol uses mouse primary chondrocytes as an example, showing the morphological characteristics and adhesion status of cells cultured for 1, 2, and 5 days after isolation. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting showed that interleukin-1β (IL-1β) treatment reduced Collagen Type II Alpha 1 (Col2a1) and increased Matrix Metallopeptidase 13 (Mmp13) expression, confirming that isolated chondrocytes respond to inflammatory stimuli. Compared with conventional methods, this protocol employs a higher collagenase concentration to reduce the impact of prolonged isolation time on cell viability, while avoiding potential cell damage from trypsin treatment. The obtained primary chondrocytes are pure and viable, suitable for in-depth studies on cartilage injury-related mechanisms, which have important implications for in vitro research and the clinical treatment of cartilage disease.

Introduction

Articular cartilage destruction is a major cause of various cartilage diseases, such as OA and RA^1,2. Chondrocytes, the predominant cell type in cartilage, are essential for maintaining homeostasis and facilitating repair in response to injury³. Alterations in chondrocyte activity not only affect the structural integrity of cartilage but also induce inflammatory responses and progressive tissue degeneration⁴. Therefore, an in-depth investigation of the biological properties of chondrocytes is crucial for elucidating the etiopathogenesis of these diseases.

Protocol

All animal experiments were approved by the Ethics Committee of Xi'an Honghui Hospital (No. 202309009), and the experimental procedures strictly adhered to the 3R principles of replacement, reduction, and refinement for animal experimentation.

Figure 1A illustrates a schematic diagram of the preparation of reagents and equipment. The Table of Materials includes information on the reagents and equipment used in this protocol.

1. Experimental mice

NOTE: Four 3-day-old, wild-type C57BL/6J mice were used in this study. 

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Results

As described in Figure 1, Figure 2, and Figure 3, we performed the protocol and successfully isolated primary chondrocytes from mouse knee joints. From approximately 10 mg of neonatal mouse cartilage, we obtained about 1 × 10⁴ cells with >90% viability. On the first day of in vitro culture, primary mouse chondrocytes exhibited a rounded morphology, with most cells in suspension and not yet ful.......

Discussion

Isolation and culture of primary chondrocytes are essential for elucidating the pathophysiology of cartilage diseases²². In primary chondrocyte culture, optimizing collagenase concentration and digestion time is crucial, as it balances the degradation of the extracellular matrix while reducing potential cellular damage¹⁷. Herein, we describe a reliable and reproducible method for isolating primary chondrocytes from mouse cartilage tissues. Using this method, approximately 1.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1× PBS Powder (2 L)	Servicebio	G0002-2L
100 mm cell culture dishes	Servicebio	CCD-100
100 μm cell sieve	Sangon Biotech	F613463-0001
15mL Conical Centrifuge Tube (Sterile, Enzyme-Free)	Servicebio	EP-1501-J
50mL Conical Centrifuge Tube (Sterile, Enzyme-Free)	Servicebio	EP-5001-J
6-well cell culture plate	Servicebio	CCP-6H
Anti-COL2A1 rabbit polyclonal antibody	Immunoway	YT1022	Primary Antibodies; WB: 1/500
Anti-GAPDH mouse monoclonal antibody	Proteintech	60004-1-IG	Primary Antibodies; WB: 1/2000
Anti-MMP13 rabbit polyclonal antibody	Immunoway	YT2796	Primary Antibodies; WB: 1/1000
Aseptic ultra-clean bench	Thermo Fisher	51029704
Cell culture incubator	Thermo Fisher	51033782
Collagenase II	Solarbio	C8150
Custom primers (Gapdh, Col2a1, Mmp13)	Sangon Biotech		Designed using Primer BLAST
DMEM/F12 medium	Hyclone	SH30023.01
Fetal bovine serum	ExCell	FCS500
HRP-Goat Anti-Mouse polyclonal antibody	Immunoway	RS0001	Secondary Antibodies; WB: 1/10000
HRP-Goat Anti-Rabbit polyclonal antibody	Immunoway	RS0002	Secondary Antibodies; WB: 1/10000
Inverted phase contrast microscope	OLYMPUS	CKX53
Micropipette	Thermo Fisher
Miniature benchtop centrifuge	SUNNE	SN-TDL-6
No. 11 surgical blade	Servicebio	QXJZ-11
Penicillin-Streptomycin Solution(100X)	Beyotime	C0222
Small Animal Dissection Instruments Set	Servicebio	QXTZ01