Preparation of vitrification and rapid warming solutions were performed according to Suzuki and colleagues with modifications described elsewhere7 (link), 8 (link). In brief, tissue was equilibrated in different solutions with varying concentrations of 10%, 20% and finally 35% ethylene glycol (Merck, Darmstadt, Germany) in GMOPS+ (Vitrolife, Gothenburg, Sweden) supplemented with 10% SSS (Serum substitute supplement, Fujifilm Irvine scientific, Santa Ana, USA) for 5 min each. The solution of 35% ethylene glycol was additionally supplemented with 5% polyvinylpyrrolidon [PVP] (Merck, Darmstadt, Germany) and 0.5 mol/L sucrose (Merck, Darmstadt, Germany). Subsequently, surplus solution was removed with sterile cellulose material and the tissue was fast loaded on customized metal meshes prior to immediate vertical immersion in liquid nitrogen.
Samples were rapid warmed submerging cortex strips in warming solution with decreasing sucrose gradients supplemented with 10% SSS in GMOPS+. Tissue was submerged in 0.8 mol/L sucrose for 1 min at 37 °C and equilibrated in 0.4 mol/L sucrose for 3 min. Tissue was then washed in GMOPS+ supplemented with 10% SSS for 5 min twice.
Samples were rapid warmed submerging cortex strips in warming solution with decreasing sucrose gradients supplemented with 10% SSS in GMOPS+. Tissue was submerged in 0.8 mol/L sucrose for 1 min at 37 °C and equilibrated in 0.4 mol/L sucrose for 3 min. Tissue was then washed in GMOPS+ supplemented with 10% SSS for 5 min twice.
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