Therapeutic Effects of Lyophilized Secretome derived from Corneal Mesenchymal Stem Cells on Corneal Epithelial Wound Healing

Abstract

Purpose : Application of lyophilized secretome derived from corneal Mesenchymal Stem Cells (cMSCs) has not been previously reported. In this study, we examined the therapeutic effects of lyophilized secretome in in vitro and in vivo wound healing models.

Methods : The cMSCs, human corneal-limbal epithelial cells (HCLE), HeLa cells, and Vero cells were cultured in proper media. When the cells reached 90% confluency, media was changed to MEM-α without serum and incubated for 48 hours. Supernatant was collected after centrifugation and considered as fresh secretome. To obtain freeze-dried secretomes, fresh cMSC-derived secretome snap-frozen in liquid nitrogen and transferred to lyophilizer machine for 48 hours. Each 1mL of fresh secretome yielded 10 mg of secretome in powder. Freeze-dried powder was dissolved in deionized water and filtered through a 0.2 µm filter. The cMSCs secretome was compared with HCLE, HeLa and Vero cells-derived secretomes in in vitro scratch assay. The therapeutic effects of lyophilized secretome from cMSCs were evaluated and compared with fresh cMSCs secretome both in vitro, using a scratch assay and TLR assay while the in vivo effects of cMSCs secretome was assessed using a mouse epithelial wound healing model.

Results : Both lyophilized and fresh cMSCs-derived secretome induced faster closure of wounds in an in vitro scratch assay of human corneal epithelial cells compared with the wounds treated by HCLE, HeLa, and Vero cells-derived secretome and culture media as control (82.2±6.1, 86.3 ± 7.2%, 54.1±7.6, 52.3±5.9 and 58.8±7.3, respectively, P<0.0001). Treatingstimulated macrophage cells by fresh or lyophilized cMSCs-derived secretomes significantly decreased their mRNA expression after activation of ICAM1, TLR3, IL6, IL8, and TNF alpha (P<0.0001). In vivo, following 2mm epithelial debridement wounds, the ratio of healed area was significantly higher in mice treated with topical application of fresh and lyophilized secretome compared with control group (87.6±2.7%, 86.1±4.6% and 64.7±16.8%, respectively, P<0.05).

Conclusions : Lyophilized cMSCs-derived secretome contain factors which can be potentially used to promote corneal epithelial healing and to suppress inflammation. The results of this study suggest the use of lyophilized cMSCs-derived secretome with extended storage periods as a promising non-invasive option in the treatment of corneal injuries.

This is a 2020 ARVO Annual Meeting abstract.