A controlled rate freezer (EF600-103, Asymptote, Cambridge, UK) was modified to achieve either NS or PS during cryopreservation by the addition of two modules designed to take polypropylene scintillation vials (Sigma, Z376825, 16 mm × 57 mm). One module was made of aluminum, the other of acetal (Fig. 1); these materials are good and poor conductors of heat respectively. These modules www.selleckchem.com/products/ve-821.html were fixed to the flat cooling plate of the EF600-103. Thermocouples (K type) we used to measure the temperature at the base, middle, and upper sample volume inside the vial, (0 mm, 20 mm, and 40 mm from base respectively)

the thermocouples were connected to a Pico Logger (Pico-technology). For small volume PS or NS studies, 5 ml aliquots of ELS were harvested and mixed 1:1 with a freezing solution (24% Me2SO, 76% Viaspan v/v) precooled to 4 °C, and once buy Tariquidar equilibrated (15 min), 80% of the excess CPA supernatant was removed, giving a final volume of 6 ml of 12% Me2SO,

38% Viaspan, and 50% ELS in culture medium, by volume. Icestart beads (1% w/v) (Asymptote) – sterile insoluble granules – which induce ice nucleation close to the equilibrium melting temperature of the mixture, were added and these sank by gravity to the base of the vial. These vials and the CRF were cooled to 4 °C before 5 vials (containing 6 ml each) were placed in the aluminum module, while 5 were placed into the acetal module (see Liothyronine Sodium Fig. 1). The EF600-103 was programmed to cool at 1 °C/min from 4 °C to −80 °C. The samples were held in the EF600-103 at −80 °C for 1 h after

the cooling cycle was complete, before being transferred to a −80 °C freezer for 7 days. The samples were warmed rapidly during 330 s in a 37 °C water bath until all the ice had melted (yielding an approximate warming rate of 15 °C/min). The Me2SO was diluted out of solution during a 10 min stepwise process with prepared chilled culture medium, with residual ice start granules remaining at the bottom of the tube and easily avoided during decanting. The samples were re-cultured in a 5% CO2 humidified incubator at 37 °C. To observe physical changes in the structure of the samples, CryoSEM was carried out. Samples recovered from storage at −80 °C were warmed slightly (25 s in a 37 °C water bath) to loosen the ice matrix from the container wall, allowing the bulk frozen samples to be removed rapidly and transferred onto dry ice (−78 °C) without re-warming. These were wrapped in foil and stored on dry ice before being transferred to a −80 °C freezer. Under liquid nitrogen, each sample was held in a metal bracket and split horizontally using a blade, giving a circular cross-section. This was transferred into a cryo scanning electron microscope (FEI XL30 FEGSEM with a Quorum pp2000 cryo-stage) and etched at −80 °C, before being coated in a thin layer (∼20 nm) of gold.