Method Article

Primary Isolation, Culture, and Phenotypic Verification of Rat Knee Articular Chondrocytes

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DOI:

10.3791/69509

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November 7th, 2025

 ,  ,  ,  ,  , 
1Department of Orthopedics, Longfu Hospital, 2Department of TCM Rheumatology, China-Japan Friendship Hospital, 3Beijing Key Lab for Immune-Mediated Inflammatory Diseases, China-Japan Friendship Hospital

* These authors contributed equally

In This Article

Summary

This protocol describes the isolation and culture of primary rat knee articular chondrocytes using sequential enzymatic digestion. The method yields chondrocytes with validated phenotypic characteristics, providing researchers with a reliable approach for cartilage research applications.

Abstract

Cartilage damage resulting from trauma, aging, or inflammatory conditions remains a significant clinical challenge due to the tissue's inherently limited regenerative capacity. Chondrocytes, being the sole cellular component of cartilage, are responsible for maintaining extracellular matrix structure and function, making them essential for understanding cartilage physiology, pathology, and regeneration. The current study presents a comprehensive and standardized protocol for the isolation, culture, and phenotypic verification of primary rat articular chondrocytes from the knee joint. The procedure utilizes young, suckling rats to ensure maximal cell viability and employs sequential enzymatic digestion with trypsin and collagenase type II for efficient cell extraction. The chondrocyte phenotype is verified through type II collagen immunofluorescence, flow cytometric analysis, and Interleukin (IL)-1β stimulation assays to confirm cellular identity and inflammatory responsiveness. This optimized and reproducible workflow enables the generation of high-purity chondrocyte cultures suitable for drug screening, mechanistic investigations, and tissue engineering applications, advancing cartilage biology research.

Introduction

Cartilage is a specialized type of connective tissue that performs multiple crucial functions, including providing support, cushioning, and shock absorption1. It plays a vital role in maintaining structural stability and ensuring mobility within the skeletal system2,3. Mechanical injury, degenerative changes, inflammation, or metabolic abnormalities can cause damage to cartilage4,5,6. Due to its inherent lack of blood vessels and nerves, combined with the limited proliferative capacity of chondrocytes, the repair of cartilage presents significant challenges. Diseases such as osteoarthritis (driven by degenerative changes and mechanical injury) and rheumatoid arthritis (caused by autoimmune inflammatory responses) can cause irreversible cartilage damage, greatly diminishing patients' quality of life7,8,9. As the exclusive cell type within cartilage tissue, chondrocytes are responsible for key functions such as the secretion and maintenance of the extracellular matrix, support of skeletal development, participation in joint cushioning and repair, and central regulation of cartilage growth and metabolism10,11,12.

Chondrocytes are confined within avascular matrix lacunae, resulting in severely limited capacity for their own proliferation and tissue repair13,14. The functional status of chondrocytes directly determines the health of cartilage tissue15. Chondrocyte dysfunction, caused by aging, inflammation, trauma, or genetic factors, results in decreased matrix synthesis and increased degradation16,17. This pathological process drives cartilage degeneration (such as in osteoarthritis), leading to the impairment of its essential support, cushioning, and lubrication functions. The limited regenerative capacity of cartilage is the core reason for the significant challenge in its repair and constitutes a key research area in modern orthopedics and regenerative medicine. Current research on cartilage has become a major focus in the field, with drug-based interventions, tissue engineering, and gene therapy all requiring the isolation of chondrocytes for in vitro studies18,19,20,21,22,23,24,25. Consequently, isolating and identifying chondrocytes constitutes a critical step in many experimental approaches. While immortalized chondrocyte-like cell lines such as SW1353 are used as convenient surrogates in some studies, they lack the expression of critical phenotypic markers, most notably type II collagen, which is essential for authentic chondrocyte function and matrix formation26. Therefore, primary chondrocytes remain the gold standard for research requiring physiological relevance. Compared to previously published protocols requiring multiple enzyme combinations or extensive mechanical manipulation, the simplified enzymatic digestion approach reduces complexity and processing time compared to methods requiring multiple enzyme combinations or extensive mechanical manipulation. Furthermore, the incorporation of comprehensive validation steps ensures consistent identification of functional chondrocytes and enhances reproducibility across experiments and laboratories.

Chondrocytes are characterized by their secretion of type II collagen, while under stimulation by inflammatory factors, they extensively synthesize and release matrix metalloproteinases (MMPs)27. Therefore, following chondrocyte isolation, the cells were identified via type II collagen immunofluorescence, and chondrocyte purity was assessed using flow cytometry. And stimulated with Interleukin (IL)-1β to confirm their characteristic synthesis and secretion of matrix metalloproteinases. This study describes the process of chondrocyte extraction and characterization from neonatal mice, establishing a foundation for subsequent chondrocyte-related experiments.

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Protocol

The use of animal subjects in this study was approved by the experimental animal care and welfare ethics committee of China-Japan Friendship Hospital (No. zryhyy21-21-05-16). The reagents and the equipment used are listed in the Table of Materials.

1. Experimental animals and preparatory work

NOTE: Cartilage from the joints of rats, rabbits, and humans following total knee arthroplasty can be used for chondrocyte isolation. However, due to their higher viability, suckling rats were utilized for chondrocyte extraction in this study.

  1. Transport suckling rats from the experimental animal production facility to the laboratory using specialized transfer containers.
  2. Euthanize suckling rats by CO2 asphyxiation (following institutionally approved protocols) and rinse their body surfaces with sterile solution.
  3. Immerse the euthanized suckling rats in 75% ethanol for 10 min to disinfect the surface contaminants.
  4. Transfer the disinfected suckling rats to a biosafety cabinet and place them in sterile Petri dishes.
    NOTE: Thorough surface cleaning and ethanol immersion of suckling rats are critical steps to prevent bacterial or fungal contamination, which may adversely affect subsequent experiments.

2. Isolation of articular cartilage tissue

NOTE: The neonatal cartilage appears as a translucent, bluish-white layer covering the bony epiphysis. Strict aseptic techniques must be followed during this step to avoid contamination that may compromise subsequent experiments.

  1. Isolate the suckling rat knee joint using a scalpel and ophthalmic scissors, then remove the overlying skin tissue.
  2. Transfer the dissected knee joint to a new sterile Petri dish and replace the scalpel and ophthalmic scissors.
  3. Harvest articular cartilage from the knee joint using a scalpel, then trim away surrounding connective tissue thoroughly with ophthalmic scissors.

3. Digestion of cartilage tissue

NOTE: Use trypsin to digest connective tissues surrounding the cartilage, followed by 0.2% collagenase II for cartilage matrix digestion.

  1. Add the harvested cartilage to 3 volumes of 0.25% trypsin and incubate in a 37 °C thermostatic shaker with agitation for 30 min.
  2. Terminate the digestion by adding PBS containing 10% fetal bovine serum (FBS). Remove adherent tissues from the cartilage using ophthalmic scissors. Centrifuge (4 °C, 500 × g, 5 min) and discard the supernatant.
  3. Resuspend the cartilage tissue in PBS, centrifuge (4 °C, 500 × g, 5 min), and discard the supernatant. Repeat this washing step 3 times.
  4. Add 0.2% collagenase II to the processed cartilage tissue and digest in a 37 °C thermostatic shaker with agitation for 4 h.

4. Chondrocyte isolation and culture

NOTE: All procedures must be performed under sterile conditions in a biosafety cabinet. Sterile gloves should be worn during mechanical dissociation of cartilage tissue using a syringe plunger on the cell strainer to prevent experimental contamination. Under standard culture conditions, primary chondrocytes typically require approximately 2-3 days to reach 80%-90% confluence for the first passage.

  1. Place a 200-µm cell strainer over a sterile culture dish and filter the digested cartilage suspension through the mesh.
  2. Grind the residual cartilage fragments on the strainer using a sterile syringe plunger, then rinse the strainer with complete DMEM medium.
  3. Collect all filtrates, centrifuge (4 °C, 500 × g, 5 min), and discard the supernatant.
  4. Resuspend the pellet in PBS, centrifuge (4 °C, 500 × g, 5 min), and discard the supernatant.
  5. Resuspend cells in complete DMEM medium and transfer to a T25 culture flask.
  6. Incubate the flask in a humidified incubator (37 °C, 5% CO2).
  7. Passage cells at this confluence using trypsin-EDTA and subculture at a 1:2 ratio.

5. Type II collagen immunofluorescence staining for chondrocyte identification

NOTE: The adhered chondrocytes are characterized by a spindle-shaped and polygonal morphology. All biomarker analyses presented in this study were performed using cells at Passage 2. Chondrocytes at passage 3 begin to exhibit more pronounced dedifferentiation, characterized by a shift toward a spindle-shaped, fibroblast-like morphology and a deceleration in proliferation rate. This phenotypic change becomes more evident after passage 4. By passage 6, the vast majority of cells adopt an elongated, fibroblast-like shape. The antibody dilutions were determined based on the manufacturer's recommendation and validated by our preliminary experiments to ensure optimal specificity and signal intensity.

  1. Seed chondrocytes in a 24-well plate and culture for 24 h to allow cell adhesion.
  2. Discard the culture medium and wash the cells 3 times with PBS (add PBS to each well, incubate for 5 min, then aspirate).
  3. Fix the cells with 4% paraformaldehyde for 10 min, then wash 3 times with PBS.
  4. Permeabilize the cells with 0.3% Triton X-100 (prepared in PBS) for 10 min.
  5. Wash the cells 3 times with PBS, then block with animal-free blocking solution (150 µL/well, diluted 5× in PBS) at room temperature for 1 h.
  6. Remove the blocking solution and incubate with primary antibody (anti-Col II, 1:200 dilution) overnight at 4 °C.
  7. Retrieve the primary antibody and wash the cells 3 times with PBS.
  8. Add Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (1:50 dilution) and incubate at 37 °C for 1.5 h in the dark.
  9. Remove the secondary antibody and wash the cells 3 times with PBS.
  10. Mount with DAPI-containing antifade medium and dry in the dark for 10 min.
  11. Observe and capture images using an inverted fluorescence microscope.

6. Flow cytometric analysis of chondrocyte purity

NOTE: Chondrocytes are characterized by their ability to synthesize and secrete type II collagen, which serves as a definitive marker for identification. The chondrosarcoma cell line SW1353, while exhibiting some chondrocytic characteristics, lacks type II collagen secretion capacity and thus serves as the negative control in this experiment.

  1. Digest both chondrocytes and SW1353 cells using EDTA-free trypsin.
  2. Neutralize digestion with complete DMEM medium and resuspend cells in PBS.
  3. Incubate cells with fixation/permeabilization solution for 15 min, then add 1 mL wash buffer.
  4. Centrifuge (500 × g, 5 min), discard the supernatant, and incubate with 100 µL anti-type II collagen antibody (1:100 dilution) for 15 min protected from light, followed by adding 1 mL wash buffer.
  5. Centrifuge (500 × g, 5 min), discard the supernatant, and incubate with Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody for 30 min protected from light, then add 1 mL wash buffer.
  6. Centrifuge (500 × g, 5 min), discard the supernatant, resuspend each sample in 200 µL wash buffer, and analyze by flow cytometry.

7. IL-1β stimulation of chondrocytes

NOTE: The production of matrix metalloproteinases (MMPs) in response to IL-1β stimulation is a hallmark feature of chondrocytes. Therefore, this study employs IL-1β stimulation to validate this phenotypic characteristic in isolated chondrocytes.

  1. Digest chondrocytes from culture flasks and resuspend in complete DMEM medium.
  2. Count the cells and adjust the concentration to 5 × 105 cells/mL using complete DMEM.
  3. Seed cells into a 6-well plate (24 wells total), with 12 wells designated as the blank control group and 12 wells as the IL-1β stimulation group.
  4. Incubate the plate in a cell culture incubator for 12 h.
  5. Add 10 ng/mL IL-1β to the stimulation group after complete cell adhesion.
  6. Collect cell culture supernatants after 24 h for Enzyme-Linked Immunosorbent Assay(ELISA) analysis.
  7. Wash the cells twice with PBS.
  8. Lyse 6 control wells and 6 IL-1β-stimulated wells with RIPA buffer for subsequent Western blot analysis.
  9. Extract RNA from 6 control wells and 6 IL-1β-stimulated wells using TRIzol for qPCR analysis.

8. Quantitative Real-Time PCR (qRT-PCR) analysis

NOTE: All RNA-related experiments were performed using DEPC-treated consumables. Briefly, tubes and tips were soaked in 0.1% DEPC for 12h, autoclaved (121 °C, 20 min) to inactivate both RNases and residual DEPC, and oven-dried prior to use.

  1. Add 1 mL Trizol to the cell pellet and homogenize using a homogenizer. Incubate at room temperature for 5 min.
  2. Add 0.2 mL chloroform and vortex vigorously for 30 s. Incubate at room temperature for 3 min.
  3. Centrifuge at 4 °C, 12,000 × g for 15 min. Transfer the colorless aqueous phase to a new microcentrifuge tube and add an equal volume of isopropanol.
  4. Mix well and incubate at room temperature for 10 min. Centrifuge at 4 °C, 12,000 × g for 10 min. Discard the supernatant and retain the RNA pellet.
  5. Add 1 mL of pre-chilled 75% ethanol to the RNA pellet. Vortex thoroughly and centrifuge at 4 °C, 12,000 × g for 5 min. Discard the supernatant and retain the RNA pellet.
  6. Air-dry the RNA pellet in a biosafety cabinet. Dissolve the RNA in 20 µL of RNase-free water.
  7. Measure the RNA concentration and quality using a Nanodrop spectrophotometer.
  8. Reverse transcribe the total RNA into cDNA using a reverse transcription kit.
  9. Prepare the reaction mixture containing 1 µg total RNA, 4 µL magnesium chloride solution, 2 µL dNTP mixture, 0.5 µL RNase inhibitor, 0.6 µL AMV reverse transcriptase, 1 µL random primers, and RNase-free water to adjust the final volume to 20 µL.
  10. Incubate the reaction at 42 °C for 15 min, followed by 95 °C for 5 min. Cool to 4 °C for 5 min, then dilute the cDNA with RNase-free water.
  11. Prepare the PCR reaction mixture containing 2 µL cDNA, 10 µL SYBR Green Real-time PCR Master Mix, forward and reverse primers (Table 1) at a final concentration of 0.2 µmol/L each, and RNase-free water to a final volume of 20 µL.
  12. Set the PCR conditions as follows: pre-denaturation at 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 45 s. Collect fluorescence data during the 72 °C extension step.
  13. After the reaction, record the CT values for each sample and the internal reference. Calculate the relative expression levels of target genes using the 2-ΔΔCT method.

9. ELISA assay

NOTE: Prepare biotinylated antibody working solution and enzyme conjugate working solution fresh before use.

  1. Remove the ELISA kit from the refrigerator 30 min in advance and allow it to equilibrate to room temperature.
  2. Prepare all required solutions according to the kit instructions.
  3. Add 100 µL of samples or standard solutions (with varying concentrations) into the designated wells (zero wells contain only standard/sample dilution buffer). Seal the plate with adhesive film and incubate at 37 °C for 90 min (exclude blank control wells).
  4. Discard the liquid and wash each well with 350 µL wash buffer. Incubate for 30 s, discard the liquid, and blot dry on thick absorbent paper. Repeat the wash 4 times.
  5. Add 100 µL of biotinylated antibody working solution to each well. Seal the plate and incubate at 37 °C for 60 min (exclude blank control wells).
  6. Discard the liquid and wash each well 4 times as described in step 4.
  7. Add 100 µL of enzyme conjugate working solution to each well. Seal the plate and incubate at 37 °C for 30 min (exclude blank control wells).
  8. Discard the liquid and wash each well 4 times as described in step 4.
  9. Add 100 µL of substrate solution to each well. Protect from light and incubate at 37 °C for 15 min.
  10. Add 100 µL of stop solution to each well, mix thoroughly, and measure the optical density (OD) at 450 nm immediately.
  11. Generate a standard curve using CurveExpert software and calculate the sample concentrations.

10. Western blot analysis

NOTE: Prior to the experiment, prepare electrophoresis buffer, transfer buffer, TBST, 5% skim milk, and cut PVDF membrane to the appropriate size in advance. Subsequently, capture the chemiluminescent signals using a chemiluminescence imaging system. Set the instrument to automatic or manual mode for gradient exposure, which typically ranges from 10 s to 5 min, to ensure signals are captured within the linear dynamic range and to avoid pixel saturation. After signal capture, select the exposure image where the target bands are clear and the background is low for subsequent quantitative analysis.

  1. Prepare RIPA lysis buffer (containing 1% PMSF and 1% phosphatase inhibitor) and add RIPA lysis buffer at a ratio of 250 µL per well of a 6-well plate.
  2. Lyse cells using an ultrasonic disruptor (3 cycles, 7-10 s each) and incubate on ice for 30 min.
  3. Centrifuge the lysate at 4 °C,12,000 × g for 10 min, then collect the supernatant for protein quantification.
  4. Determine protein concentration using the Bicinchoninic acid (BCA) assay.
  5. Adjust protein concentrations to the same level using RIPA lysis buffer.
  6. Add 5× Loading Buffer to samples at a 4:1 ratio (sample: buffer).
  7. Boil samples for 10 min, then centrifuge at 4 °C, 12,000 × g for 10 min, and collect the supernatant for electrophoresis.
  8. Assemble the SDS-PAGE gel in the electrophoresis chamber and fill with electrophoresis buffer until the comb is submerged.
  9. Remove the comb, load samples into wells, and add protein ladder to reference lanes.
  10. Run electrophoresis until the bromophenol blue dye reaches the bottom of the gel, then disassemble the apparatus and retrieve the gel.
  11. Assemble the transfer "sandwich" and perform wet transfer at constant voltage.
  12. Block the PVDF membrane with 5% skim milk in TBST for 1 h at room temperature.
  13. Incubate with diluted primary antibody overnight at 4 °C.
  14. Recover the primary antibody and wash the membrane with TBST (5 × 5 min).
  15. Incubate with HRP-conjugated secondary antibody for 90 min at room temperature.
  16. Recover the secondary antibody and wash the membrane with TBST (5 × 5 min).
  17. Prepare ECL substrate solution and store it protected from light.
  18. Apply the ECL substrate to the membrane and capture chemiluminescent signals using an imaging system with gradient exposure.
  19. Analyze band intensities using ImageJ software and calculate relative protein expression levels for further analysis.

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Results

Immunofluorescence staining for type II collagen demonstrated that the isolated chondrocytes were strongly positive (Figure 1A). Flow cytometric analysis further demonstrated that more than 98% of the cells were positive for type II collagen (Figure 1B), whereas the negative control SW1353 cells were negative (Figure 1C). Following stimulation with 10 ng/ml IL-1β for 24 h, Western blotting demonstrated a marked elevation in MMP3 and...

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Discussion

The protocol described here offers several key advantages for the isolation and culture of primary rat articular chondrocytes. The use of suckling rats is critical for achieving optimal cell yield and viability, as cartilage from young animals contains more proliferative chondrocytes and less mineralized matrix compared to adult cartilage. This age selection significantly improves the efficiency of enzymatic digestion and subsequent cell recovery.

The sequential enzymatic digestion approach re...

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Disclosures

The authors report no conflicts of interest in this work.

Acknowledgements

This study was supported by the National High-Level Hospital Clinical Research Funding and Elite Medical Professionals Initiative of China-Japan Friendship Hospital (NO.ZRJY2025-QM05).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
4% ParaformaldehydeSolarbioP1110
5× Loading BufferSolarbioP1040
Alexa Fluor 488-conjugated anti-rabbit IgGZhongshan Jingqiao BiotechnologyZF-0511
Animal-free Blocking SolutionCell Signaling Technology15019
Anti-Collagen II antibodyAbcamab34712
Bicinchoninic Acid (BCA) Protein Assay KitSolarbioPC0020
Collagenase IIYeasen Biotechnology40508ES60
DAPI-containing Fluorescent Mounting MediumZhongshan Jingqiao BiotechnologyZLI-9556
Dulbecco's Modified Eagle MediumSolarbio11995
Fetal bovine serum (FBS)SolarbioS9010
Horseradish Peroxidase (HRP)-Conjugated Goat Anti-Rabbit IgGZhongshan Jingqiao BiotechnologyZB-2301
IL-1βpeprotech400-01B
MMP13 Rabbit mAbABclonalA11148
MMP3 Rabbit mAbABclonalA11418
Non-Fat Dry MilkSolarbioD3840
Phosphate Buffered Saline (PBS)SolarbioP1020
PVDF MembraneThermo Fisher Scientific88518
Rat MMP-13 ELISA KitNouvsNBP3-06931
Rat MMP-3 ELISA KitNouvsNBP3-06894
Reverse Transcription KitPromegaA3500
RIPA Lysis BufferSolarbioR0010
RNase-free WaterSolarbioR1600
SDS-PAGE GelEpizymePG112
SYBR Green Real-time PCR Master MixToyoboQPK-201
Triton X-100SolarbioT8200
TRIzolSolarbio15596026
TrypsinSolarbioT1320
Ultra-sensitive ECL Chemiluminescence Substrate KitNCM BiotechP10300
β-Actin Rabbit mAbABclonalAC026

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Chondrocyte IsolationChondrocyte CultureRat Knee CartilageEnzymatic DigestionCollagenase Type IIFlow CytometryType II CollagenImmunofluorescence Assay

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