title: CRYOPRESERVATION STRATEGY FOR TISSUE ENGINEERING CONSTRUCTS CONSISTING OF HUMAN MESENHYMAL STEM CELLS AND HYDROGEL BIOMATERIALS.
abstract: BACKGROUND
  The development of vitrification strategy for cell-biomaterial constructs, particularly biologically inspired nanoscale materials and hydrogels mimicking the in vivo environment is an active area. A cryopreservation strategy mimicking the in vivo environment for cell-hydrogel constructs may enhance cell proliferation and biological function.
  
  
  OBJECTIVE
  To demonstrate the efficacy of vitrification as a platform technology involving tissue engineering and human mesenchymal stem cells (hMSCs).
  
  
  METHODS
  Microcarriers made from alginate coated with chitosan and collagen are used. Conventional freezing and vitrification were compared. The vitrification strategy includes 10 min step-wise exposure to a vitrification solution (40% v/v EG, 0.6M sucrose) and immersion into liquid nitrogen.
  
  
  RESULTS
  Confocal imaging of live/dead staining of hMSCs cultured on the surface of microcarriers demonstrated that vitrified cells had excellent appearance and prolonged spindle shape morphology. The proliferation ability of post-vitrified cells arbitrated to protein Ki-67 gene expression was not significantly different in comparison to untreated control, while that of post-freezing cells was almost lost. The ability of hMSCs cultured on the surface of microcarriers to proliferate has been not affected by vitrification and it was significantly better after vitrification than after conventional freezing during continuous culture. Collagen II related mRNA expression by 4 weeks post-vitrification and post-freezing showed that ability to differentiate into cartilage was sustained during vitrification and reduced during conventional freezing. No significant difference was found between control and vitrification groups only.
  
  
  CONCLUSIONS
  Vitrification strategy coupled with advances in hMSC-expansion platform that completely preserves the ability of stem cells to proliferate and subsequently differentiate allows not only to reach a critical cell number, but also demonstrate prospects for effective utilization and transportation of cells with their support system, creating demand for novel biodegradable materials.
authors.affiliation: Tissue Engineering Program; Department of Orthopaedic Surgery, YLL School of Medicine, National University of Singapore, Singapore.
authors.forename: Y
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Low Temperature Preservation Unit, National University Medical Institutes, YLL School of Medicine, National University of Singapore, Singapore.
authors.forename: F
authors.initials: F
authors.lastname: Wen
authors.affiliation: Low Temperature Preservation Unit, National University Medical Institutes, YLL School of Medicine, National University of Singapore, Singapore.
authors.forename: S S
authors.initials: SS
authors.lastname: Gouk
authors.affiliation: Tissue Engineering Program; Department of Orthopaedic Surgery, YLL School of Medicine, National University of Singapore, Singapore.
authors.forename: E H
authors.initials: EH
authors.lastname: Lee
authors.affiliation: Low Temperature Preservation Unit, National University Medical Institutes, YLL School of Medicine, National University of Singapore, Singapore. .kuleshova@imp.uni-hannover.de.
authors.forename: L
authors.initials: L
authors.lastname: Kuleshova
mesh_terms: D000464:Alginates / Q000737:chemistry; D001672:Biocompatible Materials / Q000737:chemistry*; D002356:Cartilage / Q000166:cytology*; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D048271:Chitosan / Q000737:chemistry; D003094:Collagen / Q000737:chemistry; D015925:Cryopreservation / Q000379:methods*; D005615:Freezing; D020723:Glucuronic Acid / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D006801:Humans; D020100:Hydrogels / Q000737:chemistry*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods*; D058989:Vitrification
chemical_list: D000464:Alginates; D001672:Biocompatible Materials; D006603:Hexuronic Acids; D020100:Hydrogels; D020723:Glucuronic Acid; D003094:Collagen; D048271:Chitosan