Mesenchymal Stem Cell (MSC) Aggregate Formation in vivo

Materials and Reagents

1. Human bone marrow mesenchymal stem cells expressing green fluorescent protein (GFP-MSCs) from The Center for the Preparation and Distribution of Adult Stem Cells (http://medicine.tamhsc.edu/irm/msc-distribution.html)
   
2. C57BL/6J or BALB/C mice (2-3 months of age) (The Jackson Laboratory)
   
3. Phosphate-buffered saline (PBS) without Ca²⁺ and Mg²⁺ (pH 7.4) (Life Technologies, catalog number: 10010 )
   
4. Hank’s Balanced Salt Solution (HBSS) without Ca²⁺ and Mg²⁺ (Lonza, catalog number: 04-315Q )
   
5. 0.25% trypsin with 1x EDTA (Life Technologies, catalog number: 25200 )
   
6. Minimum Essential Medium alpha (Life Technologies, catalog number: 12561 )
   
7. Premium select fetal bovine serum (Atlanta Biologicals, catalog number: S11550 )
   
8. Penicillin-streptomycin (Life Technologies, catalog number: 15140 )
   
9. 100x L-glutamine (Life Technologies, catalog number: 25030 )
   
10. Complete culture medium (CCM) for MSC growth (see Recipes)
    

Equipment

1. Stericup-GP 0.22 µm vacuum filtration device (EMD Millipore, catalog number: SCGPU05RE )
   
2. Water bath set to 37 °C
   
3. Centrifuge with swinging-bucket rotor and adaptors for 50 ml conical tubes
   
4. 50 ml sterile conical tube (BD Biosciences, Falcon^®, catalog number: 352070 )
   
5. Humidified cell culture incubator set to 37 °C and 5% CO₂
   
6. Upright microscope with 4x and 10x objectives and a filter set to visualize GFP
   
7. 29 gauge needle with 1 ml syringe (Terumo Europe N.V., catalog number: 05M2913 )
   
8. Isoflurane anesthesia system with nose cone for mouse
   
9. Sterile dissecting scissors, pins, and curved forceps with a serrated edge
   
10. Rubber or styrofoam platform
    
11. Dissecting microscope with optional camera and monitor (Figure 1)
    
12. Illumatool Bright Lights Systems LT 9900 with epi-fluorescence attachment (Lightools Research) and GFP filter set (Figure 1)
    
    
    Figure 1. Equipment required to visualize and collect GFP-MSC aggregates/spheres from the mouse peritoneum. GFP-MSC aggregates/spheres can be visualized in the mouse peritoneum using a dissecting microscope with an epi-fluorescence attachment and GFP filter set. High quality images can be acquired with an appropriate camera mounted to the dissecting scope (a camera is not a requirement for collecting the aggregates).
    

Procedure