I want to perform an in vitro wound healing assay using immortalized human corneal epithelial cells (hCEC) and observe the effect of mesenchymal stem cells (MSC) on wound healing by culturing them in culture inserts (transwell) above the epithelial cells.

How should I set up the experiment? Should I seed both cultures separately a few days before the experiment—each in its own medium—then create a scratch at the point of epithelial cell confluence and subsequently place the transwells with MSCs over the plated hCECs? Or should I seed both cell types at the same time, one above the other, from the start?

Additionally, which medium should I use for co-culture? Currently, I am using a commercial medium for corneal epithelial cells that came with the purchased cells, and DMEM/F12 with FBS and penicillin/streptomycin for MSCs.

I would greatly appreciate any advice. Thank you!