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

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 system^2,3. Mechanical injury, degenerative changes, inflammation, or metabolic abnormalities can cause damage to cartilage^4,5,6. 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 life^7,8,9. 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 metabolism^10,11,12.

Chondrocytes are confined within avascular matrix lacunae, resulting in severely limited capacity for their own proliferation and tissue repair^13,14. 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 degradation^16,17. 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 studies^{18,19,20,21,22,23,24,25}. 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.

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.

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 MMP13 protein levels in chondrocytes (Figure 2A), RT-PCR analysis revealed a significant increase in MMP3 and MMP13 mRNA expression in chondrocytes (Figure 2B) and ELISA detected a substantial rise in MMP3 and MMP13 protein content in the cell culture supernatants (Figure 2C).

Figure 1: Immunofluorescence and flow cytometry analysis of type II collagen expression. (A) Immunofluorescence detection of type II collagen in chondrocytes, with DAPI staining for nuclei. Scale bars: 500 µm. (B) Flow cytometry analysis of type II collagen labeling in chondrocytes. (C) Flow cytometry analysis of type II collagen labeling in SW1353 cells. Please click here to view a larger version of this figure.

Figure 2: IL-1β-induced upregulation of MMP3 and MMP13 in chondrocytes. Chondrocytes were stimulated with 10 ng/mL IL-1β for 24 h. (A) Western blot analysis of MMP3 and MMP13 protein expression in chondrocytes. (B) RT-PCR analysis of MMP3 and MMP13 mRNA levels in chondrocytes. (C) ELISA quantification of MMP3 and MMP13 concentrations in the culture supernatant. Data are presented as mean ± SD (n = 3). Please click here to view a larger version of this figure.

Table 1: Polymerase chain reaction (PCR) primer sequences used in this study.

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 represents a crucial aspect of this protocol. The initial trypsin treatment effectively removes peripheral connective tissues and partially disrupts the cartilage matrix, while the subsequent collagenase II digestion specifically targets the type II collagen-rich extracellular matrix surrounding chondrocytes. The 4-h collagenase digestion time has been optimized to balance complete cell release with minimal cell damage. Shorter digestion periods may result in incomplete cell liberation, while extended exposure can compromise cell viability and phenotype.

Mechanical dissociation using a syringe plunger is essential for breaking apart cartilage fragments and releasing individual cells. This step must be performed gently to avoid excessive cell damage while ensuring adequate tissue disruption. The use of sterile techniques throughout the procedure is paramount, as chondrocyte cultures are particularly susceptible to contamination.

The multi-faceted validation approach employed in this protocol ensures the authenticity and functionality of isolated chondrocytes. Type II collagen immunofluorescence staining serves as the gold standard for chondrocyte identification, as this protein is exclusively produced by chondrocytes in cartilaginous tissues²⁸. The high purity achieved (>98% positive cells) demonstrates the effectiveness of the isolation method and minimal contamination with other cell types, such as synoviocytes or fibroblasts.

Flow cytometric analysis provides a quantitative assessment of cell purity and can be used for high-throughput quality control²⁹. The use of SW1353 cells as a negative control is particularly valuable. Although SW1353 cells exhibit chondrocyte-like properties and are commonly used as a surrogate for chondrocytes in experiments, they do not express type II collagen, a key characteristic of functional chondrocytes³⁰. This makes them a reliable negative reference in such studies.

The IL-1β stimulation assay confirmed that isolated chondrocytes retained their functional capacity to react to inflammatory signals. IL-1β treatment triggered MMP13 upregulation at both transcriptional and secretory levels, verifying the cells' pathological response and affirming their utility for disease modeling research. It should be noted that this response is a hallmark yet non-exclusive feature of chondrocytes, as other joint-associated cells like fibroblast-like synoviocytes and mesenchymal stem cells also upregulate MMPs upon IL-1β stimulation.

Compared to previously published protocols, this method offers several improvements. The simplified enzymatic approach reduces the complexity and time requirements compared to methods requiring multiple enzyme combinations or extensive mechanical manipulation. The incorporation of comprehensive validation steps ensures consistency and reproducibility across different experiments and laboratories.

The protocol's emphasis on maintaining sterile conditions throughout the procedure minimizes contamination risks, which have been a significant challenge in chondrocyte culture. Additionally, the detailed troubleshooting guidelines and quality control measures help ensure successful outcomes even for novice researchers. The high-quality chondrocytes obtained using our isolation and culture protocol hold significant potential for various bioengineering applications. Primarily, these cells serve as an essential cell source for cartilage tissue engineering, where they can be seeded onto biocompatible scaffolds to create functional constructs for repairing articular cartilage defects^31,32. Furthermore, with the advancement of 3D bioprinting technology, these chondrocytes can be utilized as bio-inks to fabricate sophisticated, patient-specific cartilage tissue models for drug screening and disease modeling³³.

Limitations and considerations
Despite its advantages, this protocol has certain limitations that should be acknowledged. The use of young animals limits the direct relevance to age-related cartilage diseases, where aged chondrocytes may exhibit different biological characteristics. Researchers studying age-related cartilage degeneration may need to adapt this protocol for older animals, though this typically results in lower cell yields and altered cell behavior. Chondrocytes isolated using this method are best used within the first three passages, as extended culture periods can lead to dedifferentiation and loss of the chondrocytic phenotype. Researchers should monitor cell morphology and marker expression throughout culture to ensure maintenance of the desired phenotype.