title: Development of a novel vitrification method for chondrocyte sheets.
abstract: BACKGROUND
  There is considerable interest in using cell sheets for the treatment of various lesions as part of regenerative medicine therapy. Cell sheets can be prepared in temperature-responsive culture dishes and applied to injured tissue. For example, cartilage-derived cell sheets are currently under preclinical testing for use in treatment of knee cartilage injuries. The additional use of cryopreservation technology could increase the range and practicality of cell sheet therapies. To date, however, cryopreservation of cell sheets has proved impractical.
  
  
  RESULTS
  Here we have developed a novel and effective method for cryopreserving fragile chondrocyte sheets. We modified the vitrification method previously developed for cryopreservation of mammalian embryos to vitrify a cell sheet through use of a minimum volume of vitrification solution containing 20% dimethyl sulfoxide, 20% ethylene glycol, 0.5 M sucrose, and 10% carboxylated poly-L-lysine. The principal feature of our method is the coating of the cell sheet with a viscous vitrification solution containing permeable and non-permeable cryoprotectants prior to vitrification in liquid nitrogen vapor. This method prevented fracturing of the fragile cell sheet even after vitrification and rewarming. Both the macro- and microstructures of the vitrified cell sheets were maintained without damage or loss of major components. Cell survival in the vitrified sheets was comparable to that in non-vitrified samples.
  
  
  CONCLUSIONS
  We have shown here that it is feasible to vitrify chondrocyte cell sheets and that these sheets retain their normal characteristics upon thawing. The availability of a practical cryopreservation method should make a significant contribution to the effectiveness and range of applications of cell sheet therapy.
authors.forename: Miki
authors.initials: M
authors.lastname: Maehara
authors.forename: Masato
authors.initials: M
authors.lastname: Sato
authors.forename: Masahito
authors.initials: M
authors.lastname: Watanabe
authors.forename: Hitomi
authors.initials: H
authors.lastname: Matsunari
authors.forename: Mami
authors.initials: M
authors.lastname: Kokubo
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kanai
authors.forename: Michio
authors.initials: M
authors.lastname: Sato
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Matsumura
authors.forename: Suong-Hyu
authors.initials: SH
authors.lastname: Hyon
authors.forename: Munetaka
authors.initials: M
authors.lastname: Yokoyama
authors.forename: Joji
authors.initials: J
authors.lastname: Mochida
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nagashima
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002470:Cell Survival; D019902:Chondrocytes / Q000166:cytology*; D015925:Cryopreservation / Q000379:methods*; D004622:Embryo, Mammalian; D011817:Rabbits; D058989:Vitrification