The Comparison of MSC Secretome Effects on Corneal Wound Healing Depending on the Cell Culture Environments

The therapeutic effects of secreted factors (secretome) produced by human mesenchymal stem cells (MSCs) were evaluated depending on the cell culture environments. The cells were cultured on standard 2D culture conditions and 3D electrospun fiber scaffolds, and the secretomes were collected. The goal was to determine whether culturing MSCs within a 3D, extracellular matrix-like environment would improve the therapeutic.

The secretome of MSCs cultured on 2D substrates and 3D electrospun fiber scaffolds showed substantial compositional differences. The relative amounts of factors such as HGF and ICAM-1 were increased over 5 times in 3D cultures compared to 2D cultures. Cell viability of corneal fibroblasts sustained metabolic activity for 6 days when co-cultured with MSCs grown on the electrospun fiber scaffolds in the absence of serum. A scratch-based wound healing assay showed 95% closure at 48 hours in corneal fibroblast monolayers co-cultured with MSCs grown on electrospun fibers, while the control group only exhibited 50% closure at the same time point. When electrospun fibers with seeded MSCs were applied to an ex vivo rabbit corneal organ culture system, it promoted re-epithelialization. Immunostaining of the corneas showed that the expression of alpha-SMA was lower compared to the injured cornea without treatment. In addition, keratan sulfate expression was higher in the treated group compared to the untreated.

MSCs cultured on three-dimensional, electrospun fiber scaffolds facilitate wound healing effects on corneal fibroblasts in vitro and on explanted corneas in an organ culture model through differential secretome profiles compared to MSCs cultured on 2D substrates.