MSC Seeded Scaffold Preparation: A Technique to Prepare Scaffold Seeded with Mesenchymal Stem Cells for Surgical Implantation into Murine Model

1. Cell Culture and Scaffold Preparation

NOTE: Scaffolds should be prepared 48 h prior to implantation in mice. The following volumes are provided on a per-scaffold basis. Multiply quantities as needed for additional scaffolds.

1. Cut poly(lactic acid) (PLA) scaffolds into resection cavity-sized (approximately 2 mm x 2 mm) pieces. Scaffolds can be cut by hand using scissors or using a hole punch for repeatability.
2. Sterilize by immersing in 70% ethanol for 15 min followed by immersing in PBS. Place scaffold in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin while preparing cells for seeding.
3. Lift cultured MSCs using 0.05% trypsin (3–5 mL for a T75 flask). Incubate at 37 °C for 5 min. Ensure that the cells have lifted, then add 7–10 mL DMEM to the flask to inactivate trypsin.
4. Transfer flask contents to a 15 mL centrifuge tube. Count cells using a hemocytometer. Pellet 5 x 10⁵ MSCs for each scaffold via centrifugation at 100 x g for 5 min.
5. Aspirate off supernatant and resuspend MSCs in 5 µL DMEM per 5 x 10⁵ cells.
6. Prepare the scaffolds for seeding by patting them dry.
   1. Remove a scaffold from DMEM immersion with forceps and temporarily place it on the lid of the 6-well plate. Lift the scaffold off the lid, leaving behind a droplet of excess DMEM.
   2. Place the scaffold back on the lid again in a new, dry location. Repeat 3–5 times, then place the partially dried scaffold in a new 6-well plate for seeding. Repeat for each scaffold.
      NOTE: A partially dried scaffold provides optimal cell seeding results. If the scaffold is too wet, cells will slide off the scaffold and adhere to the well plate below. If the scaffold is too dry, the droplet will not spread over the entire scaffold, resulting in poor initial cell distribution.
7. Using a pipette, gently mix the vial of stem cells to homogenize the suspension as some cells may have settled to the bottom.
8. Slowly pipette 2.5 µL freshly mixed MSC suspension directly on the scaffold, creating a small droplet over the top of the scaffold.
9. Add 300 µL DMEM to edges of each well. This will prevent rapid evaporation of the cell droplet. Incubate at 37 °C for 30 min, allowing the cells to attach to the scaffold.
10. With forceps, gently flip the scaffolds over in the well plate. Seed 2.5 µL freshly mixed MSC suspension (this results in a total of 5 x 10⁵ cells seeded per scaffold). Incubate at 37 °C for 30 min, allowing the cells to attach to the scaffold.
11. Cover the scaffolds in DMEM by adding 2 mL to each well of the 6-well plate. Gently lift the scaffolds to allow media to flow underneath them. Incubate at 37 °C in this state for 48 h prior to implantation surgery.
12. Check the scaffolds after 24 h and add/change media if excess evaporation or discolored media due to pH imbalance is observed.