title: Isolation of equine multipotent mesenchymal stromal cells by enzymatic tissue digestion or explant technique: comparison of cellular properties.
abstract: BACKGROUND
  The treatment of tendon lesions with multipotent mesenchymal stromal cells (MSCs) is widely used in equine medicine. Cell sources of MSCs include bone marrow, as well as solid tissues such as adipose tissue. MSCs can be isolated from these solid tissues either by enzymatic digestion or by explant technique. However, the different preparation techniques may potentially influence the properties of the isolated MSCs. Therefore, the aim of this study was to investigate and compare the effects of these two different methods used to isolate MSCs from solid tissues.Equine adipose tissue, tendon and umbilical cord matrix served as solid tissue sources of MSCs with different stiffness and density. Subsequent to tissue harvest, MSCs were isolated either by enzymatic digestion with collagenase or by explant technique. Cell yield, growth, differentiation potential and tendon marker expression were analysed.
  
  
  RESULTS
  At first passage, the MSC yield was significantly higher in enzymatically digested tissue samples than in explanted tissue samples, despite a shorter period of time in primary culture. Further analysis of cell proliferation, migration and differentiation revealed no significant differences between MSCs isolated by enzymatic digestion and MSCs isolated by explant technique. Interestingly, analysis of gene expression of tendon markers revealed a significantly higher expression level of scleraxis in MSCs isolated by enzymatic digestion.
  
  
  CONCLUSIONS
  Both isolation techniques are feasible methods for successful isolation of MSCs from solid tissues, with no major effects on cellular proliferation, migration or differentiation characteristics. However, higher MSC yields were achieved in a shorter period of time by collagenase digestion, which is advantageous for the therapeutic use of MSCs. Moreover, based on the higher level of expression of scleraxis in MSCs isolated by enzymatic digestion, these cells might be a better choice when attempting tendon regeneration.
authors.affiliation: Large Animal Clinic for Surgery, University of Leipzig, An den Tierkliniken 21, 04103 Leipzig, Germany. claudia.gittel@vetmed.uni-leipzig.de.
authors.forename: Claudia
authors.initials: C
authors.lastname: Gittel
authors.forename: Walter
authors.initials: W
authors.lastname: Brehm
authors.forename: Janina
authors.initials: J
authors.lastname: Burk
authors.forename: Henriette
authors.initials: H
authors.lastname: Juelke
authors.forename: Carsten
authors.initials: C
authors.lastname: Staszyk
authors.forename: Iris
authors.initials: I
authors.lastname: Ribitsch
mesh_terms: D050156:Adipogenesis / Q000502:physiology; D000273:Adipose Tissue / Q000166:cytology; D000818:Animals; D002452:Cell Count / Q000662:veterinary; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000502:physiology; D017364:Collagenases; D003584:Cytological Techniques / Q000379:methods / Q000662:veterinary; D015870:Gene Expression / Q000502:physiology; D006736:Horses; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D010012:Osteogenesis / Q000502:physiology; D059748:Proteolysis; D013710:Tendons / Q000166:cytology; D014470:Umbilical Cord / Q000166:cytology
chemical_list: D017364:Collagenases