title: Efficient recovery of undifferentiated human embryonic stem cell cryopreserved with hydroxyethyl starch, dimethyl sulphoxide and serum replacement.
abstract: BACKGROUND
  The therapeutic use of human embryonic stem cells (hESCs) is dependent on an efficient cryopreservation protocol for long-term storage. The aim of this study was to determine whether the combination of three cryoprotecting reagents using two freezing systems might improve hESC recovery rates with maintenance of hESC pluripotency properties for potential cell therapy application.
  
  
  METHODS
  Recovery rates of hESC colonies which were frozen in three cryoprotective solutions: Me2SO/HES/SR medium, Defined-medium® and Me2SO/SFB in medium solution were evaluated in ultra-slow programmable freezing system (USPF) and a slow-rate freezing system (SRF). The hESC pluripotency properties after freezing-thawing were evaluated.
  
  
  RESULTS
  We estimated the distribution frequency of survival colonies and observed that independent of the freezing system used (USPF or SRF) the best results were obtained with Me2SO/HES/SR as cryopreservation medium. We showed a significant hESC recovery colonies rate after thawing in Me2SO/HES/SR medium were 3.88 and 2.9 in USPF and SRF, respectively. The recovery colonies rate with Defined-medium® were 1.05 and 1.07 however in classical Me2SO medium were 0.5 and 0.86 in USPF and SRF, respectively. We showed significant difference between Me2SO/HES/SR medium×Defined-medium® and between Me2SO/HES/SR medium×Me2SO medium, for two cryopreservation systems (P<0.05).
  
  
  CONCLUSIONS
  We developed an in house protocol using the combination of Me2SO/HES/SR medium and ultra-slow programmable freezing system which resulted in hESC colonies that remain undifferentiated, maintain their in vitro and in vivo pluripotency properties and genetic stability. This approach may be suitable for cell therapy studies.
authors: Hemotherapy Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Brazil; Department of Clinical Medicine, Ribeirão Preto Medical School, University of São Paulo, Brazil. Electronic address: mdelgado@hemocentro.fmrp.usp.br.
authors: Maristela Delgado
authors: MD
authors: Orellana
authors: Hemotherapy Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Brazil.
authors: Gil Cunha
authors: GC
authors: De Santis
authors: Department of Pediatrics, Ribeirão Preto Medical School, University of São Paulo, Brazil.
authors: Kuruvilla Joseph
authors: KJ
authors: Abraham
authors: Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, Brazil.
authors: Aparecida Maria
authors: AM
authors: Fontes
authors: Hemotherapy Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Brazil.
authors: Danielle Aparecida Rosa
authors: DA
authors: Magalhães
authors: Department of Dental Materials and Prosthodontics, School of Dentistry of Ribeirão Preto, University of São Paulo, Brazil.
authors: Viviane de Cássia
authors: Vde C
authors: Oliveira
authors: Hemotherapy Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Brazil.
authors: Everton de Brito Oliveira
authors: Ede B
authors: Costa
authors: Hemotherapy Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Brazil.
authors: Patrícia Vianna Bonini
authors: PV
authors: Palma
authors: Hemotherapy Center of Ribeirão Preto, Ribeirão Preto Medical School, University of São Paulo, Brazil; Department of Clinical Medicine, Ribeirão Preto Medical School, University of São Paulo, Brazil.
authors: Dimas Tadeu
authors: DT
authors: Covas
mesh_terms: D002470:Cell Survival / Q000187:drug effects; D064987:Cell- and Tissue-Based Therapy / Q000379:methods; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D005615:Freezing; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D006895:Hydroxyethyl Starch Derivatives / Q000494:pharmacology*; D010952:Plasma Substitutes / Q000494:pharmacology*; D039904:Pluripotent Stem Cells / Q000166:cytology
chemical_list: D003451:Cryoprotective Agents; D016895:Culture Media, Serum-Free; D006895:Hydroxyethyl Starch Derivatives; D010952:Plasma Substitutes; D004121:Dimethyl Sulfoxide
keywords: Cryopreservation; Dimethyl sulphoxide (Me2SO); Human embryonic stem cells; Hydroxyethyl starch (HES); Pluripotency capacity; Serum Replacement (SR); hESC colony recovery rate