INTRODUCTION:
The secretion of trophic factors by MSCs has gained increased interest given the benefits it may bring to the treatment of a variety of traumatic injuries such as skin wounds. Herein, we report on a 3D culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived MSCs (UCX®) towards the application of conditioned medium for the treatment of cutaneous wounds.
METHODS:
An UCX® 3D culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of ECM proteins and trophic factors involved in the wound healing process was analysed. The skin regenerative potential of UCX® 3D-culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® 2D-culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively.
RESULTS:
UCX® spheroids kept in our 3D system remained viable and multipotent and secreted considerable amounts of VEGF-A and FGF-2, undetected in 2D cultures, and higher amounts of MMP-2, MMP-9, HGF, TGF-β1, G-CSF, FGF-2 and IL-6, when compared to CM2D. Furthermore, CM3D significantly enhanced elastin production and migration of keratinocytes and fibroblasts in vitro. In turn, tubulogenesis assays revealed increased capillary maturation in the presence of CM3D, as seen by significant increase in capillary thickness and length, when compared to CM2D; and increased branching points and capillary number, when compared to basal medium. Finally, CM3D-treated wounds presented signs of faster and better resolution when compared to untreated and CM2D-treated wounds in vivo. Although CM2D proved to be beneficial, CM3D-treated wounds revealed a completely regenerated tissue by day 14 after excisions, with a more mature vascular system and already showing glands and hair follicles.
CONCLUSIONS:
This work unravels an important alternative to the use of cells in the final formulation of advanced therapy medicinal products (ATMPs) by providing a proof of concept that a reproducible system for the production of UCX® conditioned medium can be used to prime a secretome for eventual clinical applications.