title: Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions.
abstract: OBJECTIVE
  Mesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues.
  
  
  METHODS
  MSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays.
  
  
  RESULTS
  Post-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure.
  
  
  CONCLUSIONS
  A method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.
authors.affiliation: National Institute of Science and Technology in Stem Cell and Cell Therapy, Center for Cell Therapy and Regional Blood Center, Ribeirao Preto, Brazil. karen@hemocentro.fmrp.usp.br
authors.forename: Karen
authors.initials: K
authors.lastname: de Lima Prata
authors.forename: Gil Cunha
authors.initials: GC
authors.lastname: de Santis
authors.forename: Maristela Delgado
authors.initials: MD
authors.lastname: Orellana
authors.forename: Patricia Vianna Bonini
authors.initials: PV
authors.lastname: Palma
authors.forename: María Sol
authors.initials: MS
authors.lastname: Brassesco
authors.forename: Dimas Tadeu
authors.initials: DT
authors.lastname: Covas
mesh_terms: D017667:Adipocytes / Q000166:cytology; D000818:Animals; D002454:Cell Differentiation; D049109:Cell Proliferation; D048430:Cell Shape; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D020732:Cytogenetic Analysis; D006801:Humans; D016130:Immunophenotyping; D007231:Infant, Newborn; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010011:Osteocytes / Q000166:cytology; D014470:Umbilical Cord / Q000166:cytology*; D015262:Xenobiotics / Q000032:analysis
chemical_list: D015262:Xenobiotics