Three-dimensional spheroid cell culture of umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced paracrine induction of wound healing

Introduction: The secretion of trophic factors by mesenchymal stromal cells 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 three-dimensional culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived mesenchymal stromal cells (UCX (R)) towards the application of conditioned medium for the treatment of cutaneous wounds. Methods: A UCX (R) three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX (R) spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX (R) three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX (R) two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively. Results: UCX (R) spheroids kept in our three-dimensional system remained viable and multipotent and secreted considerable amounts of vascular endothelial growth factor A, which was undetected in two-dimensional cultures, and higher amounts of matrix metalloproteinase-2, matrix metalloproteinase-9, hepatocyte growth factor, transforming growth factor beta 1, granulocyte-colony stimulating factor, fibroblast growth factor 2 and interleukin-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 a 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 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 by providing a proof of concept that a reproducible system for the production of UCX (R)-conditioned medium can be used to prime a secretome for eventual clinical applications.