title: Optimizing isolation culture and freezing methods to preserve Wharton's jelly's mesenchymal stem cell (MSC) properties: an MSC banking protocol validation for the Hellenic Cord Blood Bank.
abstract: BACKGROUND
  Mesenchymal stem or stromal cells (MSCs) are a heterogeneous population that can be isolated from many tissues including umbilical cord Wharton's jelly (UC-WJ). Although initially limited in studies such as a hematopoietic stem cell transplantation adjuvant, an increasing number of clinical trials consider MSCs as a potential anti-inflammatory or a regenerative medicine agent. It has been proposed that creating a repository of MSCs would increase their availability for clinical applications. The aim of this study was to assess the optimal isolation and cryopreservation procedures to facilitate WJ MSC banking.
  
  
  METHODS
  Cells were isolated from UC-WJ using enzymatic digestion or plastic adhesion methods. Their isolation efficacy, growth kinetics, immunophenotype, and differentiation potential were studied, as well as the effects of freezing. Flow cytometry for common MSC markers was performed on all cases and differentiation was shown with histocytochemical staining. Finally, the isolation efficacy on cryopreserved WJ tissue fragments was tested.
  
  
  RESULTS
  MSC isolation was successful using both isolation methods on fresh UC-WJ tissue. However, UC-WJ MSC isolation from frozen tissue fragments was impossible. Flow cytometry analysis revealed that only MSC markers were expressed on the surface of the isolated cells while differentiation assays showed that they were capable of trilinear differentiation. All the above characteristics were also preserved in isolated UC-WJ MSCs over the cryopreservation study period.
  
  
  CONCLUSIONS
  These data showed that viable MSCs can only be isolated from fresh UC-WJ tissue, setting the foundation for clinical-grade banking.
authors.affiliation: Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens, Athens, Greece.
authors.forename: Theofanis K
authors.initials: TK
authors.lastname: Chatzistamatiou
authors.forename: Andreas C
authors.initials: AC
authors.lastname: Papassavas
authors.forename: Efstathios
authors.initials: E
authors.lastname: Michalopoulos
authors.forename: Christos
authors.initials: C
authors.lastname: Gamaloutsos
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Mallis
authors.forename: Ioanna
authors.initials: I
authors.lastname: Gontika
authors.forename: Effrosyni
authors.initials: E
authors.lastname: Panagouli
authors.forename: Stauros L
authors.initials: SL
authors.lastname: Koussoulakos
authors.forename: Catherine
authors.initials: C
authors.lastname: Stavropoulos-Giokas
mesh_terms: D064591:Allografts; D001771:Blood Banks; D018929:Cell Culture Techniques / Q000379:methods* / Q000592:standards; D002469:Cell Separation / Q000379:methods* / Q000592:standards; D005260:Female; D005312:Fetal Blood; D006801:Humans; D008297:Male; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D011309:Preservation, Biological / Q000379:methods* / Q000592:standards