title: Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks.
abstract: BACKGROUND
  In vitro fabricated tissue engineered vascular constructs could provide an alternative to conventional substitutes. A crucial factor for tissue engineering of vascular constructs is an appropriate cell source. Vascular cells from the human umbilical cord can be directly isolated and cryopreserved until needed. Currently no cell bank for human vascular cells is available. Therefore, the establishment of a future human vascular cell bank conforming to good manufacturing practice (GMP) conditions is desirable for therapeutic applications such as tissue engineered cardiovascular constructs.
  
  
  METHODS
  A fundamental step was the adaption of conventional research and development starting materials to GMP compliant starting materials. Human umbilical cord artery derived cells (HUCAC) and human umbilical vein endothelial cells (HUVEC) were isolated, cultivated, cryopreserved (short- and long-term) directly after primary culture and recultivated subsequently. Cell viability, expression of cellular markers and proliferation potential of fresh and cryopreserved cells were studied using trypan blue staining, flow cytometry analysis, immunofluorescence staining and proliferation assays. Statistical analyses were performed using Student's t-test.
  
  
  RESULTS
  Sufficient numbers of isolated cells with acceptable viabilities and homogenous expression of cellular markers confirmed that the isolation procedure was successful using GMP compliant starting materials. The influence of cryopreservation was marginal, because cryopreserved cells mostly maintain phenotypic and functional characteristics similar to those of fresh cells. Phenotypic studies revealed that fresh cultivated and cryopreserved HUCAC were positive for alpha smooth muscle actin, CD90, CD105, CD73, CD29, CD44, CD166 and negative for smoothelin. HUVEC expressed CD31, CD146, CD105 and CD144 but not alpha smooth muscle actin. Functional analysis demonstrated acceptable viability and sufficient proliferation properties of cryopreserved HUCAC and HUVEC.
  
  
  CONCLUSIONS
  Adaptation of cell isolation, cultivation and cryopreservation to GMP compliant starting materials was successful. Cryopreservation did not influence cell properties with lasting impact, confirming that the application of vascular cells from the human umbilical cord is feasible for cell banking. A specific cellular marker expression profile was established for HUCAC and HUVEC using flow cytometry analysis, applicable as a GMP compliant quality control. Use of these cells for the future fabrication of advanced therapy medicinal products GMP conditions are required by the regulatory authority.
authors.affiliation: German Heart Institute Berlin, Department of Cardiothoracic and Vascular Surgery, Laboratory for Tissue Engineering, Augustenburger Platz 1, 13353 Berlin, Germany.
authors.forename: Bianca
authors.initials: B
authors.lastname: Polchow
authors.forename: Kati
authors.initials: K
authors.lastname: Kebbel
authors.forename: Gerno
authors.initials: G
authors.lastname: Schmiedeknecht
authors.forename: Anne
authors.initials: A
authors.lastname: Reichardt
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Henrich
authors.forename: Roland
authors.initials: R
authors.lastname: Hetzer
authors.forename: Cora
authors.initials: C
authors.lastname: Lueders
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D003114:Colony-Forming Units Assay; D015925:Cryopreservation / Q000379:methods* / Q000592:standards*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology*; D019983:Guideline Adherence; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology; D014015:Tissue Banks / Q000592:standards*; D014469:Umbilical Arteries / Q000166:cytology*
chemical_list: D015415:Biomarkers; D016326:Extracellular Matrix Proteins