title: [Comparative studies on different cryopreservation protocols of human amniotic fluid-derived mesenchymal stem cells].
abstract: OBJECTIVE
  To isolate and culture human amniotic fluid-derived mesenchymal stem cells (HAFMSCs), to investigate a better cryopreservation protocol of HAFMSCs and to observe the bio-characteristics and the multi-potential of HAFMSCs after cryopreservation for the further fundamental researches and clinical applications.
  
  
  METHODS
  HAFMSCs were isolated from the amniotic fluid of pregnant women during the second trimester by the improved two-step method. HAFMSCs were cryopreserved with different cryopreservation protocols (containing different contents of FBS and DMSO at cryoprotectant) in liquid nitrogen for 12 weeks. The bio-characteristics of the HAFMSCs after cryopreservation were analyzed. The growth characteristics were observed by MTT method and the growth curves were drawn. The surface antigens of HAFMSCs were detected using flow cytometry, including CD29, CD34, CD44, CD45, CD73, and CD90. The adipogenic and osteogenic differentiation abilities of HAFMSCs were observed. The mRNA levels of Oct-4 and Nanog of the HAFMSCs were compared between before and after cryopreservation.
  
  
  RESULTS
  At 12 weeks after cryopreservation, different protocols had different effects on the cell viability; the better formula of cryoprotectant was 50% DMEM, 40% FBS, and 10% DMSO. After cryopreservation, the cells proliferated rapidly and the growth curves showed "S" shape, which was the same as the cells before cryopreservation. Phenotype showed that HAFMSCs were positive for the surface markers CD29, CD44, CD73, and CD90, and negative for CD34 and CD45. After 21 days of adipogenic differentiation, the lipid droplets were observed by oil red O staining. After 21 days of osteogenic differentiation, the calcium mineralization were verified by von Kossa staining. There was no significant difference (P > 0.05) in the mRNA levels of Oct-4 and Nanog between before and after cryopreservation.
  
  
  CONCLUSIONS
  HAFMSCs have rapid proliferation and multi-potential in vitro. The cells have high viabilities and no changes of the bio-characteristics and differentiation potentialities after cryopreservation for 12 weeks. Cryoprotectant containing 50% DMEM, 40% FBS, and 10% DMSO is a better cryopreservation protocol.
authors.affiliation: Institute of Geriatric Cardiology, General Hospital of Chinese PLA, Beijing, 100853, PR China.
authors.forename: Yiru
authors.initials: Y
authors.lastname: Wang
authors.forename: Jing
authors.initials: J
authors.lastname: Bai
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.forename: Lifeng
authors.initials: L
authors.lastname: Liu
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000653:Amniotic Fluid / Q000166:cytology*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000737:chemistry; D005260:Female; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D011247:Pregnancy
chemical_list: D003451:Cryoprotective Agents