In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

Procedures involving animal subjects were approved by the local Institutional Animal Care and Ethics Committee, with an effort to minimize animal suffering. The following protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at Shanghai Ninth People’s Hospital affiliated to Shanghai JiaoTong University School of Medicine with the protocol number [2017]063.

1. Establishment of a Surgically-Induced Rat Knee Osteoarthritis Model

1. For this surgical procedure, use 8 – 12 week old male Sprague Dawley (SD) rats with a body weight between 250 and 300 g.
2. Anesthetize the animals with 40 mg/kg tiletamine and 40 mg/kg zolazepam via intramuscular injection.
3. Place the animal in a left lateral position on a heated stage. Shave the right knee thoroughly with a razor. Disinfect the surgical area with 10% providone-iodine solution followed by 70% ethanol, repeat two more times and then paint with 10% providone-iodine solution and cover the non-surgical area with a surgical pad.
4. Using a surgical scalpel, make a 2 cm incision laterally along the patellar tendon to expose the jointcapsules.
5. Use a surgical scalpel to transect the medial collateral ligament. Reflect the meniscus toward the femur.
6. Drip sterile 0.9% sodium chloride solution on the surface of articular cartilage during the operation to prevent the cartilage from drying out.
7. Grab the medial meniscus with forceps and cut through the meniscus at its narrowest point from the tibial attachment with a scalpel (Figure 1A). Avoid injuring the cartilage.
8. Close the joint capsule with a 4 – 0 absorbable suture.
9. Monitor the surgical rats until they regain consciousness.
10. Inject 20 mg/kg gentamicin to prevent infection and 0.05 mg/kg buprenorphine through an intramuscular route to relieve pain after surgery.
11. Maintain the rats in a temperature-controlled animal facility under a 12 h light/dark cycle for 3 – 8 weeks to onset KOA. Animals continue to receive pain medication, including but not limited to Buprenorphine (0.05 mg/kg, i.m.) and Gentamicin (20 mg/kg, i.m.), if pain symptoms persist.

2. Histology Assessment of Rat KOA Model

1. Sacrifice the rats with excessive CO₂ inhalation (Add a fill rate of about 30% of the chamber volume per minute with carbon dioxide to the existing air in the chamber to make the animal unconsciousness within 2-3 min. Maintain CO2 flow for a minimum of 1 min after respiration ceases.) at indicated time points (representative results are shown at 8 weeks of KOA induction ranging from 3 to 8 weeks) after the surgery. 
2. Dissect the skin and muscle around the knee joints and cut off the knee joints with a scalpel.
3. Fix the knee joints in 4% paraformaldehyde (PFA) for 3 days at room temperature. Afterwards, decalcify the samples in 20% ethylenediaminetetraacetic acid (EDTA) for about 4 weeks at 4 °C, and change the EDTA every 3 days.
   Caution: Paraformaldehyde is toxic, wear appropriate personal protective equipment (PPE), such as gloves and eyeglasses.
4. Dehydrate the samples using an automatic tissue processor. Set the menu to start a flush cycle as follows: 70% ethanol for 1 h; then 80% ethanol and 95% ethanol for 1 h respectively; followed by 2 cycles of 100% ethanol for 1 h; subsequently, xylene twice for 1 h each time and paraffin 3 times at 58 °C for 1 h each time.
   Caution: Xylene is toxic, so wear appropriate PPE, such as gloves and eyeglasses.
   NOTE: Optionally, use Coplin Jars for manual dehydration with the same set-up as the automatic tissue processor.
5. Place the medial aspect of the intact joint face down and embed it into the paraffin block with tissue embedded into the center.
   NOTE: Optionally, use forceps to embed the samples into the paraffin block manually.
6. Start to cut 5 µm sagittal sections from the medial margin of the joint for 30 µm intervals with 2 serial sections by rotary microtome.
7. Mount sections on glass slides and deparaffinize the slides in xylene. Between the 2 serial sections, stain one slide with Hematoxylin & Eosin (H&E) staining: 0.1% Hematoxylin for 15 min, 1% Acetic Acid Solution for 10 s and 0.5% Eosin for 10 min. And stain the other slide with Safranin O/Fast Green staining: 0.1% Hematoxylin for 15 min, 0.02% Fast Green for 3 min, 1% Acetic Acid Solution for 10 s and 0.1% Safranin O for 3 min.
   Caution: Xylene is toxic, wear appropriate PPE, such as gloves, eyeglasses.
8. Score the stained slides using Osteoarthritis Research Society International (OARSI) Histopathology Assessment System in rats⁶.

3. Labelling of Human Mesenchymal Stem Cells with the Fluorescent Dye DiD

1. Prepare DiD cell-labeling solution in 100% ethanol at a concentration of 1 mM. Protect the cell-labeling solution from light and store it at room temperature up for 6 months.
2. Grow haMSCs with standard cell culture procedures in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% penicillin and streptomycin and 10% fetal calf serum (FCS) in a 10 cm petri dish.
3. Detach haMSCs with 2 mL 0.25% trypsin with 1 mM EDTA prior to around 80% confluence.
4. Neutralize trypsin with 4 mL culture medium and spin the cells at 800 x g for 3 min at room temperature.
5. Suspend the cells at a density of 1 x 10⁶ cells/mL in 5 mL serum-free culture medium.
6. Label the cells by adding 50 µL DiD cell-labeling solution in 5 mL serum-free culture medium at 37 °C for 50 min.
7. Spin the labeled cells at 800 x g for 3 min.
8. Wash the cells two more times with 5 mL phosphate buffer saline (PBS) at 800 x g for 3 min each.
9. Check the labelled haMSCs using a fluorescent microscope with a Cy5 filter set.
10. Count the cells with trypan blue staining and obtain 2.5 x 10⁶ viable cells in 100 µL PBS for injection.

4. In Vivo Fluorescent Imaging to Track haMSCs

1. Eight weeks after the surgery (Step 1), anesthetize the KOA rats by intramuscular injection of 25 mg/kg tiletamine and 25 mg/kg zolazepam.
2. Shave the right knee thoroughly with a razor to expose the joint area. Disinfect the joint area with 70% ethanol.
3. Using a 26 G syringe needle, inject 2.5 x 10⁶ labeled cells suspended in 100 µL PBS at the center of the triangle area formed by the medial side of the patellar ligament, the medial femoral condyle, and the medial tibial condyle (Figure 1B).
4. Open the imaging software and initialize the in vivo bioluminescence imaging system.
5. Position rats in the supine position in the central stage of the imaging system and close the door of the chamber.
6. Check the "fluorescent" checkbox and select fluorescent filter sets of 640 nm for excitation, and 680 nm for emission (Figure 2A).
7. Select field of view to D. Set pixel binning to medium (8), F/Stop to 2 and exposure time to auto. Maintain the optimal exposure time consistent to obtain comparable results across the entire study (Figure 2A).
8. Remove the animal from the stage and monitor for recovery from anesthesia.
   NOTE: Place animal on an heating pad covered with 2-3 layers of toweling.
9. Choose the units of radiant efficiency for the measurement of fluorescence (Figure 2B). Select regions of interest (ROI) for analysis and quantify fluorescent signals from each image (Figure 2C).
10. Repeat the procedure of in vivo imaging for each rat sequentially to study haMSCs longitudinal retention in the joint.
    NOTE: We suggest imaging the animals' fluorescent signals by in vivo imaging system once every two days for the first week after injection and once every week until the signal cannot be detected.