MSC to chondrocyte mitochondrial transfer quantification

Phase 1: Cell Culture & Preparation (Day 0)

• Thaw cryopreserved human bone marrow-derived MSCs (Lonza, cat# PT-2501) and immortalized human chondrocytes (TC28a2 line) according to manufacturer's protocols
• Culture MSCs in MSCGM-CD BulletKit medium (Lonza, cat# PT-3001) at 37°C, 5% CO₂ on collagen I-coated flasks (Corning, cat# 354471)
• Culture chondrocytes in DMEM/F-12 (Gibco, cat# 11330) supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C, 5% CO₂
• Passage MSCs at 80% confluence (use cells P3-P5); passage chondrocytes at 80% confluence (use cells P10-P15)
• Prior to co-culture, label MSCs with MitoTracker Green FM (Thermo Fisher, cat# M7514) at 200 nM in serum-free medium for 30 min at 37°C, then wash 3× with PBS
• Label chondrocytes with MitoTra

Phase 1: Cell Culture & Preparation (Day 0)

• Thaw cryopreserved human bone marrow-derived MSCs (Lonza, cat# PT-2501) and immortalized human chondrocytes (TC28a2 line) according to manufacturer's protocols
• Culture MSCs in MSCGM-CD BulletKit medium (Lonza, cat# PT-3001) at 37°C, 5% CO₂ on collagen I-coated flasks (Corning, cat# 354471)
• Culture chondrocytes in DMEM/F-12 (Gibco, cat# 11330) supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C, 5% CO₂
• Passage MSCs at 80% confluence (use cells P3-P5); passage chondrocytes at 80% confluence (use cells P10-P15)
• Prior to co-culture, label MSCs with MitoTracker Green FM (Thermo Fisher, cat# M7514) at 200 nM in serum-free medium for 30 min at 37°C, then wash 3× with PBS
• Label chondrocytes with MitoTracker Red CMXRos (Thermo Fisher, cat# M7512) at 100 nM in serum-free medium for 30 min at 37°C, then wash 3× with PBS
• Validate mitochondrial labeling efficiency by imaging ( Zeiss Axio Observer) and flow cytometry (≥95% positive required)

Phase 2: Co-Culture Setup (Day 0-1)

• Prepare transwell inserts (Corning, cat# 3460) with 0.4 μm pores allowing only soluble factor/mitochondrial vesicle passage
• Seed 1×10⁵ labeled chondrocytes in the bottom chamber of a 12-well plate in chondrocyte culture medium
• Seed 1×10⁵ MitoTracker Green-labeled MSCs in the transwell insert (MSC:chondrocyte ratio = 1:1)
• Include controls: chondrocytes alone (no MSC), MSCs alone (no chondrocytes), and chondrocytes with 10 μM Cytochalasin D (Sigma, cat# C2618) to inhibit tunneling nanotube formation
• Incubate at 37°C, 5% CO₂ for 24 hours for baseline mitochondrial transfer

Phase 3: Time Course Assessment (Day 1-7)

• At each timepoint, harvest chondrocytes by trypsinization (0.25% EDTA-free trypsin, Gibco, cat# 12605) for 5 min at 37°C
• Centrifuge cells at 300×g for 5 min, resuspend in 200 μL FACS buffer (PBS + 2% FBS + 2 mM EDTA)
• Fix cells with 4% paraformaldehyde (Electron Microscopy Sciences, cat# 15710) for 15 min at room temperature
• Permeabilize with 0.1% Triton X-100 (Sigma, cat# X100) for 10 min for intracellular staining
• Stain with anti-Tomm20-PE antibody (BD Biosciences, cat# 612278) at 1:50 dilution for mitochondrial identification
• Include single-color controls for compensation: MitoTracker Green alone, MitoTracker Red alone, Tomm20-PE alone, and unstained cells

Phase 4: Flow Cytometry Acquisition (Day 1-7)

• Use BD LSRFortessa X-20 flow cytometer with 488 nm (Blue) and 561 nm (Yellow-Green) lasers
• Acquire 50,000 events per sample at low flow rate (400 events/sec max)
• Gating strategy:

• Analyze using FlowJo v10.8 software
• Calculate transfer efficiency as: (% double-positive chondrocytes / total chondrocytes) × 100

Phase 5: Osteoarthritis Model Validation (Parallel Arms)

• In separate wells, treat chondrocytes with 5 ng/mL IL-1β (R&D Systems, cat# 201-LB) + 10 ng/mL TNF-α (R&D Systems, cat# 210-TA) for 24h to simulate OA inflammatory environment
• Compare mitochondrial transfer rates in OA-like conditions vs baseline
• Measure intracellular ROS using CellROX Deep Red (Thermo Fisher, cat# C10422) at 5 μM for 30 min at 37°C
• Measure mitochondrial membrane potential using JC-1 dye (Thermo Fisher, cat# M34152) according to manufacturer's protocol

Phase 6: Functional Validation (Day 7-14)

• Assess chondrocyte survival by Annexin V/PI staining (Thermo Fisher, cat# V13241) via flow cytometry
• Measure ATP production using CellTiter-Glo 2.0 assay (Promega, cat# G9241)
• Perform Seahorse XF Real-Time ATP Rate Assay (Agilent, cat# 103592-100) to measure mitochondrial vs glycolytic ATP production
• Collect conditioned media for ELISA measurements of inflammatory cytokines (IL-6, IL-8, TNF-α; R&D Systems, cat# D6050)