title: Three-dimensional spheroid cell culture of umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced paracrine induction of wound healing.
abstract: BACKGROUND
  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®) towards the application of conditioned medium for the treatment of cutaneous wounds.
  
  
  METHODS
  A UCX® three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX® three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively.
  
  
  RESULTS
  UCX® 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 β1, 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®-conditioned medium can be used to prime a secretome for eventual clinical applications.
authors.affiliation: ECBio - Investigação e Desenvolvimento em Biotecnologia S.A., Rua Henrique Paiva Couceiro, N° 27, 2700-451, Amadora, Portugal. miguel.santos@ecbio.com.
authors.forename: Jorge M
authors.initials: JM
authors.lastname: Santos
authors.affiliation: iMed.ULisboa - Research Institute for Medicines, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal. sergio.jp.camoes@gmail.com.
authors.forename: Sérgio P
authors.initials: SP
authors.lastname: Camões
authors.affiliation: iMed.ULisboa - Research Institute for Medicines, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal. elyssecf@hotmail.com.
authors.forename: Elysse
authors.initials: E
authors.lastname: Filipe
authors.affiliation: iMed.ULisboa - Research Institute for Medicines, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal. mzrfcipriano@ff.ulisboa.pt.
authors.forename: Madalena
authors.initials: M
authors.lastname: Cipriano
authors.affiliation: ECBio - Investigação e Desenvolvimento em Biotecnologia S.A., Rua Henrique Paiva Couceiro, N° 27, 2700-451, Amadora, Portugal. rita.barcia@ecbio.com.
authors.forename: Rita N
authors.initials: RN
authors.lastname: Barcia
authors.affiliation: ECBio - Investigação e Desenvolvimento em Biotecnologia S.A., Rua Henrique Paiva Couceiro, N° 27, 2700-451, Amadora, Portugal. mariana.filipe@ecbio.com.
authors.forename: Mariana
authors.initials: M
authors.lastname: Filipe
authors.affiliation: ECBio - Investigação e Desenvolvimento em Biotecnologia S.A., Rua Henrique Paiva Couceiro, N° 27, 2700-451, Amadora, Portugal. mariana.teixeira@ecbio.com.
authors.forename: Mariana
authors.initials: M
authors.lastname: Teixeira
authors.affiliation: iMed.ULisboa - Research Institute for Medicines, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal. ssimoes@ff.ulisboa.pt.
authors.forename: Sandra
authors.initials: S
authors.lastname: Simões
authors.affiliation: iMed.ULisboa - Research Institute for Medicines, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal. mgaspar@ff.ulisboa.pt.
authors.forename: Manuela
authors.initials: M
authors.lastname: Gaspar
authors.affiliation: I3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal. dyogomask@gmail.com.|INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua do Campo Alegre, N° 823, 4150-180, Porto, Portugal. dyogomask@gmail.com.
authors.forename: Diogo
authors.initials: D
authors.lastname: Mosqueira
authors.affiliation: I3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal. dsn@ineb.up.pt.|INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua do Campo Alegre, N° 823, 4150-180, Porto, Portugal. dsn@ineb.up.pt.
authors.forename: Diana S
authors.initials: DS
authors.lastname: Nascimento
authors.affiliation: I3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal. perpetua@ineb.up.pt.|INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua do Campo Alegre, N° 823, 4150-180, Porto, Portugal. perpetua@ineb.up.pt.|ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira, N° 228, 4050-313, Porto, Portugal. perpetua@ineb.up.pt.|Unit for Lymphopoiesis, Immunology Department, INSERM U668, University Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Institut Pasteur, Paris, 75015, France. perpetua@ineb.up.pt.
authors.forename: Perpétua
authors.initials: P
authors.lastname: Pinto-do-Ó
authors.affiliation: ECBio - Investigação e Desenvolvimento em Biotecnologia S.A., Rua Henrique Paiva Couceiro, N° 27, 2700-451, Amadora, Portugal. pedro.cruz@ecbio.com.
authors.forename: Pedro
authors.initials: P
authors.lastname: Cruz
authors.affiliation: ECBio - Investigação e Desenvolvimento em Biotecnologia S.A., Rua Henrique Paiva Couceiro, N° 27, 2700-451, Amadora, Portugal. helder.cruz@ecbio.com.
authors.forename: Helder
authors.initials: H
authors.lastname: Cruz
authors.affiliation: iMed.ULisboa - Research Institute for Medicines, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal. mcastro@ff.ulisboa.pt.
authors.forename: Matilde
authors.initials: M
authors.lastname: Castro
authors.affiliation: iMed.ULisboa - Research Institute for Medicines, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal. jmiranda@ff.ulisboa.pt.
authors.forename: Joana P
authors.initials: JP
authors.lastname: Miranda
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D019070:Cell Lineage; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000032:analysis; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D019899:Paracrine Communication / Q000502:physiology*; D010641:Phenotype; D051381:Rats; D017208:Rats, Wistar; D014470:Umbilical Cord / Q000166:cytology; D014945:Wound Healing / Q000502:physiology*
chemical_list: D016326:Extracellular Matrix Proteins; D036341:Intercellular Signaling Peptides and Proteins