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

Prepare the scaffolds 48 hours prior to implantation in mice by first cutting PLA scaffolds into resection cavity-sized pieces of approximately 2 by 2 millimeters. Sterilize the scaffolds by immersing in 70% ethanol for 15 minutes. After 15 minutes, immerse the scaffolds in PBS. Then, place the scaffolds in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum and 1% penicillin-streptomycin while preparing cells for seeding.

To prepare the cells, first, lift cultured MSCs using 0.05% trypsin. Incubate at 37 degrees Celsius for 5 minutes. Following the incubation, ensure that the cells have lifted. Then, add DMEM to the flask to inactivate the trypsin. Next, transfer the flask contents to a 15-milliliter centrifuge tube. Count the cells using a hemocytometer. Then, pellet 5 times 10 to the fifth MSCs for each scaffold via centrifugation at 100 x g for 5 minutes.

Following the centrifugation, aspirate the supernatant and resuspend the MSCs in 5 microliters of DMEM per 5 times 10 to the fifth cells. Then, remove a scaffold from DMEM immersion with forceps and place it on the lid of the 6-well plate. Lift the scaffold off the lid, leaving behind a droplet of excess DMEM. Place the scaffold back on the lid again in a new dry location. Repeat three to five times. Then, place the partially dried scaffold in a new 6-well plate for seeding.

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 plate below. If the scaffold is to dry, the droplet will not spread over the entire scaffold, resulting in poor initial cell distribution.

Using a pipette, gently mix the tube of MSCs to homogenize the suspension as some cells may have settled to the bottom. Slowly pipette 2.5 microliters of freshly mixed MSC suspension directly on the scaffold, creating a small droplet over the top of the scaffold. Add 300 microliters of DMEM to edges of each well to prevent rapid evaporation of the cell droplet. Incubate at 37 degrees Celsius for 30 minutes, allowing the cells to attach to the scaffold.

Following the incubation, use forceps to gently flip the scaffolds over in the well plate. Seed 2.5 microliters of freshly mixed MSC suspension to give a total of 5 times 10 to the fifth cells seeded per scaffold. After an incubation of 30 minutes to allow the cells to attach to the scaffold, cover the scaffolds in DMEM by adding 2 milliliters to each well of the 6-well plate. Gently lift the scaffolds to allow media to flow underneath them. Incubate at 37 degrees Celsius in this state for 48 hours prior to implantation surgery.