Low-Temperature Storage of NPCs
The NPCs maintained in N2 medium were dissociated into small cell clumps by exposure to 0.05% trypsin and 0.04% EDTA in PBS and were neutralized in DMEM/F12 (1:1) containing 10% FBS. Cells were collected by centrifugation and transferred into a 1.2-ml cryovial (Nalge Nunc International) containing 1 ml of freezing medium (90% serum and 10% dimethyl sulfoxide [DMSO; Sigma]). Vials were slowly cooled (—FC/ min) to -800C and stored in liquid nitrogen. Thawing was in a 370C water bath, and the freezing medium was diluted gradually with 10 ml of culture medium. Cells were plated as described above or incubated in culture medium containing 0.1% trypan blue solution (Sigma) for evaluation of viability. buy avandia online
Spherical structures in expanded NPC cultures (Step 3) were vitrified by incubation for 3 min in 10% glycerol in Hepes-buffered Tyrode albumin lactate pyruvate medium containing 20% fetal bovine serum (TH20), 3 min in 10% glycerol and 20% ethylene glycol in TH20, and then briefly (30-60 sec) in 25% glycerol plus 25% ethylene glycol in TH20. Spherical structures were drawn into a serological pipette and allowed to settle toward the tip before individual drops were slowly released directly into liquid nitrogen. These solid drops were collected with precooled forceps and sealed in liquid nitrogen-filled cryovials for storage. Warming and recovery of spherical structures was performed by pouring the vitrified drops and liquid nitrogen into a container. The solid drops were quickly moved into a solution of 0.5 M sucrose/TH20 for 3 min at room temperature and then transferred to 0.25 M sucrose and 0.125 M sucrose for 3 min each at room temperature before being rinsed and cultured.